Novel Compositions for the Delivery of Negatively Charged Molecules

ABSTRACT

This invention features permeability enhancer molecules, and methods, to increase membrane permeability of various molecules, such as nucleic acids, polynucleotides, oligonucleotides, enzymatic nucleic acid molecules, antisense nucleic acid molecules, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNAs, dsRNAs, siRNAs, aptamers, or antisense nucleic acids containing nucleic acid cleaving chemical groups, peptides, polypeptides, proteins, carbohydrates, steroids, metals and small molecules, thereby facilitating cellular uptake of such molecules.

FIELD OF THE INVENTION

This patent application is a continuation of Beigelman et al., U.S. Ser.No. 09/120,520, filed Jul. 21, 1998, entitled “NOVEL COMPOSITIONS FORTHE DELIVERY OF NEGATIVELY CHARGED MOLECULES”, which claims priorityfrom Beigelman et al., U.S. Ser. No. 60/053,517, filed Jul. 23, 1997,entitled “NOVEL COMPOSITIONS FOR THE DELIVERY OF NEGATIVELY CHARGEDMOLECULES” and Beigelman et al., U.S. Ser. No. 60/072,967, filed Jan.29, 1998, entitled “NOVEL COMPOSITIONS FOR THE DELIVERY OF NEGATIVELYCHARGED MOLECULES”. These applications are hereby incorporated byreference herein in their entirety including the drawings.

BACKGROUND OF THE INVENTION

The following is a brief description of the delivery of biopolymers.This summary is not meant to be complete but is provided only forunderstanding of the invention that follows. This summary is not anadmission that all of the work described below is prior art to theclaimed invention.

Trafficking of large, charged molecules into living cells is highlyrestricted by the complex membrane systems of the cell. Specifictransporters allow the selective entry of nutrients or regulatorymolecules, while excluding most exogenous molecules such as nucleicacids and proteins. The two major strategies for improving the transportof foreign nucleic acids into cells are the use of viral vectors orcationic lipids and related cytofectins. Viral vectors can be used totransfer genes efficiently into some cell types, but they cannot be usedto introduce chemically synthesized molecules into cells. An alternativeapproach is to use delivery formulations incorporating cationic lipids,which interact with nucleic acids through one end and lipids or membranesystems through another (for a review see Felgner, 1990, Advanced DrugDelivery Reviews, 5, 162-187; Felgner 1993, J. Liposome Res., 3, 3-16).Synthetic nucleic acids as well as plasmids may be delivered using thecytofectins, although their utility is often limited by cell-typespecificity, requirement for low serum during transfection, andtoxicity.

Since the first description of liposomes in 1965, by Bangham (J. Mol.Biol. 13, 238-252), there has been a sustained interest and effort inthe area of developing lipid-based carrier systems for the delivery ofpharmaceutically active compounds. Liposomes are attractive drugcarriers since they protect biological molecules from degradation whileimproving their cellular uptake.

One of the most commonly used classes of liposomes formulations fordelivering polyanions (e.g., DNA) are those that contain cationiclipids. Lipid aggregates can be formed with macromolecules usingcationic lipids alone or including other lipids and amphiphiles such asphosphatidylethanolamine. It is well known in the art that both thecomposition of the lipid formulation as well as its method ofpreparation have effect on the structure and size of the resultantanionic macromolecule-cationic lipid aggregate. These factors can bemodulated to optimize delivery of polyanions to specific cell types invitro and in vivo. The use of cationic lipids for cellular delivery ofbiopolymers have several advantages. The encapsulation of anioniccompounds using cationic lipids is essentially quantitative due toelectrostatic interaction. In addition, it is believed that the cationiclipids interact with the negatively charged cell membranes initiatingcellular membrane transport (Akhtar et al., 1992, Trends Cell Bio., 2,139; Xu et al., 1996, Biochemistry 35, 5616).

The transmembrane movement of negatively charged molecules such asnucleic acids may therefore be markedly improved by coadministrationwith cationic lipids or other permeability enhancers (Bennett et al.,1992 Mol. Pharmacol., 41, 1023-33; Capaccioli et al., 1993, BBRC, 197,818-25; Ramila et al., 1993 J. Biol. Chem., 268, 16087-16090; Stewar etal., 1992, Human Gene Therapy, 3, 267-275). Since the introduction ofthe cationic lipid DOTMA and its liposomal formulation Lipofectin®(Felgner et al., 1987, PNAS 84, 7413-7417; Eppstein et al., U.S. Pat.No. 4,897,355), a number of other lipid-based delivery agents have beendescribed primarily for transfecting mammalian cells with plasmids orantisense molecules (Rose, U.S. Pat. No. 5,279,833; Eppand et al. U.S.Pat. No. 5,283,185; Gebeyehu et al., U.S. Pat. No. 5,334,761; Nantz etal., U.S. Pat. No. 5,527,928; Bailey et al., U.S. Pat. No. 5,552,155;Jesse, U.S. Pat. No. 5,578,475). However, each formulation is of limitedutility because it can deliver plasmids into some but not all celltypes, usually in the absence of serum (Bailey et al., 1997,Biochemistry, 36, 1628). Concentrations (charge and/or mass ratios) thatare suitable for plasmid delivery (˜5,000 to 10,000 bases in size) aregenerally not effective for oligonucleotides such as synthetic ribozymesor antisense molecules (˜10 to 50 bases). Also, recent studies indicatethat optimal delivery conditions for antisense oligonucleotides andribozymes are different, even in the same cell type. However, the numberof available delivery vehicles that may be utilized in the screeningprocedure is highly limited, and there continues to be a need to developtransporters that can enhance nucleic acid entry into many types ofcells.

Eppstein et al., U.S. Pat. No. 5,208,036, disclose a liposome,LIPOFECTIN™, that contains an amphipathic molecule having a positivelycharged choline head group (water soluble) attached to a diacyl glycerolgroup (water insoluble). Promega (Wisconsin) markets another cationiclipid, TRANSFECTAM™, which can help introduce nucleic acid into a cell.

Wagner et al., 1991, Proc. Nat. Acad. Sci. USA 88, 4255; Cotten et al.,1990, Proc. Nat. Acad. Sci. USA 87, 4033; Zenke et al., 1990, Proc. Nat.Acad. Sci. USA 87, 3655; and Wagner et al., 1990, Proc. Nat. Acad. Sci.USA 87, 3410, describe transferrin-polycation conjugates which mayenhance uptake of DNA into cells. They also describe the feature of areceptor-mediated endocytosis of transferrin-polycation conjugates tointroduce DNA into hematopoietic cells.

Wu et al., 1991, J. Biol. Chem. 266, 14338, describe in vivoreceptor-mediated gene delivery in which anasialoglycoprotein-polycation conjugate consisting of asialoorosomucoidis coupled to poly-L-lysine. A soluble DNA complex was formed capable ofspecifically targeting hepatocytes via asialoglycoprotein receptorspresent on the cells.

Clark et al., International PCT Publication No. WO 91/18012, describecell internalizable covalently bonded conjugates having an“intracellularly cleavable linkage” such as a “disulfide cleavablelinkage” or an enzyme labile ester linkage.

Brigham, U.S. Pat. No. 5,676,954 describes a method for the expressionof nucleic acid following transfection into a target organ consisting ofmammalian cells.

The references cited above are distinct from the presently claimedinvention since they do not disclose and/or contemplate the deliveryvehicles of the instant invention.

SUMMARY OF THE INVENTION

This invention features lipid-based compositions, such as cationiclipid-based compositions, that facilitate delivery of molecules into abiological system, for example into cells such as mammalian cells. Thepresent invention discloses the design, synthesis, and cellular testingof novel agents for the delivery of various molecules, for examplenucleic acids, polynucleotides, oligonucleotides, and/or negativelycharged molecules, in vitro and in vivo. Also disclosed are screeningprocedures for identifying the optimal delivery vehicles for any givennucleic acid and cell type. In general, the transporters or cytofectinsdescribed here are designed to be used either individually or as part ofa multi-component system. Examples of such multi-component systemscomprising lipid compounds of the invention are shown in Tables II, VI,VII, VIII, and IX. The lipid compounds of the invention generally shownin FIG. 1 and Table X, are expected to improve delivery of nucleicacids, polynucleotides, oligonucleotides, and/or negatively chargedmolecules, into a number of cell types originating from differenttissues, in the presence or absence of serum.

In one embodiment, the invention features a cationic lipid having theformula I:

wherein, n is 1, 2 or 3 carbon atoms; n₁ is 2, 3, 4 or 5 carbon atoms; Rand R₁ independently represent C12-C22 alkyl chain which are saturatedor unsaturated, wherein the unsaturation is represented by 1-4 doublebonds; and R₂ and R₃ are independently H, acyl, alkyl, carboxamidine,aryl, acyl, substituted carboxamidine, polyethylene glycol (PEG) or acombination thereof.

In another embodiment, the invention features a cationic lipid havingthe formula II:

wherein, n is 1, 2 or 3 carbon atoms; n₁ is 2, 3, 4 or 5 carbon atoms; Rand R₁ independently represent C12-C22 alkyl chain which are saturatedor unsaturated, wherein the unsaturation is represented by 1-4 doublebonds; and Alk represents methyl, hydroxyalkyl (e.g., hydroxymethyl andhydroxyalkyl) or a combination thereof.

In another embodiment the invention features a cationic lipid having theformula III:

wherein, R and R₁ independently represent C12-C22 alkyl chain which aresaturated or unsaturated, wherein the unsaturation is represented by 1-4double bonds; and R₂ is H, PEG, acyl or alkyl.

In another embodiment the invention features a cationic lipid having theformula IV:

wherein, n is 1-6 carbon atoms; R and R₁ independently represent C12-C22alkyl chain which are saturated or unsaturated, wherein the unsaturationis represented by 1-4 double bonds; and R₂ is H, carboxamidine, alkyl,acyl, aryl, PEG, substituted carboxamidine

Where R₃ is H, or PO₃H₂ and R₄ is OH, NH₂ or ═O.

In one embodiment the invention features a cationic lipid having theformula V:

wherein, n is 1-6 carbon atoms; X and X₁ independently represent C12-C22alkyl chain which are saturated or unsaturated, wherein the unsaturationis represented by 1-4 double bonds; B is a nucleic acid base or H; andR₅ is H, PEG, or carboxamidine.

In another embodiment the invention features a cationic lipid having theformula VI:

wherein, n is 1, 2 or 3 carbon atoms; R and R₁ independently representC12-C22 alkyl chain which are saturated or unsaturated, wherein theunsaturation is represented by 1-4 double bonds; and R₂ and R₃ isindependently H, polyethylene glycol (PEG) or

In the above referenced formulae, N, O, and H are Nitrogen, Oxygen, andHydrogen, according to the abbreviations well-known in the art.

In another embodiment the invention features a cationic lipid having theformula VII:

R₆-L₁-Cholesterol

wherein, R₆ is selected from the group consisting of arginyl methylester, arginyl amide, homoarginyl methyl ester, homoarginyl amide,ornithine methyl ester, ornithine amide, lysyl methyl ester, lysylamide, triethylenetetramine (TREN), N,N′-di-carboxamidine TREN, N-benzylhistidyl methyl ester, pyridoxyl and aminopropylimidazole. L₁ is alinker represented by R₇PO2, wherein R₇ is H, CH₃, or CH₂CH₃. Examplesof this group of compounds are: PH55933, PH55938, PH55939, PH55941,PH55942, PH55943 and PH55945.

In another embodiment the invention features a cationic lipid having theformula VIII:

R₈-L₂-Cholesterol

wherein, R₈ is selected from the group consisting of arginyl, N-Bocarginyl, homoarginyl, N-Boc homoarginyl, ornithine, N-Boc ornithine,N-benzyl histidyl, lysyl, N-Boc lysyl, N-methyl arginyl, N-methylguanidine, guanidine and pyridoxyl. L₂ is a linker represented by NH,glycine, N-butyldiamine or guanidine. Examples of this compound is Bocarginine cholesteryl amide (DS46596), N-guanyl-cholesterylamide(DS57511).

In one embodiment the invention features a cationic lipid having theformula IX:

Wherein R is independently a C12-C22 alkyl chain which are saturated orunsaturated, wherein the unsaturation is represented by 1-4 double bondsand R₁ is represented by TREN, N,N′-di-carboxamidine TREN, lysyl,arginyl, ornithyl, homoarginyl, histidyl, aminopropylimidazole, sperminecarboxylic acid.

In one embodiment, the invention features process for the synthesis ofthe compounds of formula I-IX and/or Lipid ID Nos: 700, 701, 705, 709,719, 732, 736, 737, 738, 739, 742, 743, 744, 751, 752, or 753.

In another embodiment, multi-domain cellular transport vehicles (MCTV)including one or more lipids of formula I-IX and/or Lipid ID Nos: 700,701, 705, 709, 719, 722, 723, 725, 726, 727, 732, 736, 737, 738, 739,742, 743, 744, 745, 746, 747, 749, 750, 751, 752, and/or 753, thatenhance the cellular uptake and transmembrane permeability of variousmolecules, for example, nucleic acids, polynucleotides, oligonucleotidesand/or negatively charged molecules in a variety of cell types areprovided. Examples of such lipids are shown in Table X. The lipids ofthe invention are used either alone or in combination with othercompounds with a neutral or a negative charge including but not limitedto neutral lipid and/or targeting components, to improve theeffectiveness of the lipid formulation in delivering and targetingmolecules such as nucleic acids, polynucleotides, oligonucleotides, ornegatively charged polymers to cells. In addition, these deliveryvehicles can be used to increase the transport of other impermeableand/or lipophilic compounds into cells.

In one embodiment, the invention features a compound comprising formulaI-IX and/or Lipid ID Nos: 700, 701, 705, 709, 719, 732, 736, 737, 738,739, 742, 743, 744, 751, 752, or 753. In another embodiment, theinvention features a lipid formulation comprising Formulation ID Nos:282-533.

In another embodiment, the lipid formulation further comprises atargeting component. Targeting components of the invention includeligands for cell surface receptors including, peptides and proteins,glycolipids, lipids, carbohydrates, and their synthetic variants.

In yet another embodiment, the lipid molecules of the invention, such ascationic lipids, are provided as a lipid aggregate, such as a liposome,and co-encapsulated with the compound or polymer to be delivered.Liposomes, which can be unilamellar or multilamellar, can introduceencapsulated material into a cell by different mechanisms. See, Ostro,Scientific American 102, January 1987. For example, the liposome candirectly introduce its encapsulated material into the cell cytoplasm byfusing with the cell membrane. Alternatively, the liposome can becompartmentalized into an acidic vacuole (i.e., an endosome) having a pHbelow 7.0. This low pH allows ion-pairing of the encapsulated enhancersand the negatively charged polymer, which facilitates diffusion of theenhancer:polymer complex out of the liposome, the acidic vacuole, andinto the cellular cytoplasm.

In another embodiment the invention features a lipid aggregateformulation including phosphatidylcholine (of varying chain length;e.g., egg yolk phosphatidylcholine), cholesterol, a cationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000(DSPE-PEG₂₀₀₀). The cationic lipid component of this lipid aggregate canbe any cationic lipid known in the art such as dioleoyl1,2-diacyl-3-trimethylammonium-propane (DOTAP). In yet anotherembodiment this lipid aggregate comprises a lipid described in any ofthe Formulae I-IX and/or Lipid ID Nos: 700, 701, 705, 709, 719, 722,723, 725, 726, 727, 732, 736, 737, 738, 739, 742, 743, 744, 745, 746,747, 749, 750, 751, 752, and/or 753.

In yet another embodiment, polyethylene glycol (PEG) is covalentlyattached to the lipids of the present invention. The attached PEG can beany molecular weight but is preferrably between 2000-5000 daltons.

The molecules and methods of the present invention are particularlyadvantageous for introducing nucleic acid molecules into a cell. Forexample, the invention can be used for nucleic acid delivery, such asenzymatic nucleic acid delivery, where a target site of action existsintracellularly.

In one embodiment, the invention features a lipid formulation,comprising a lipid molecule of the invention or any combination thereof,and a molecule or combination of molecules to be delivered, for example,a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide,protein, carbohydrate, steroid, polymer, metal or small molecule.

In another embodiment, the invention features a method of transfecting acell, comprising contacting a lipid formulation of the invention withthe cell under conditions suitable for the transfection.

In one embodiment, a molecule that is complexed with a lipid molecule ofthe invention includes a nucleic acid, polynucleotide, oligonucleotide,peptide, polypeptide, protein, carbohydrate, steroid, polymer, metal orsmall molecule.

In one embodiment, a nucleic acid molecule of the invention comprises anenzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5Aantisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA,siRNA, aptamer, or antisense nucleic acids containing nucleic acidcleaving chemical groups.

A molecule that is complexed with a lipid molecule of the inventionincludes an enzymatic nucleic acid, for example a hammerhead, Inozyme,G-gleaver, DNAzyme, Amberzyme, Zinzyme and/or allozyme.

In another embodiment, the invention features a method for the deliveryof molecules contemplated by the invention, such as nucleic acids,polynucleotides, oligonucleotides, peptides, proteins and/or negativelycharged polymers, into cells such as suspension cells and T cells usingcytofectins of the present invention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will be first described briefly.

DRAWINGS

FIGS. 1A-C depict the different classes of lipids of the instantinvention.

FIG. 2 depicts a scheme for the synthesis of diaminobutyric andguanidinium-based cationic lipids.

FIG. 3 depicts a scheme for the synthesis of Boc Arginine cholesterylamide (12; DS46596).

FIG. 4 depicts a scheme for the synthesis of cholesterol-lysine-methylester-methylphosphonoamidate (PH55933; 15) andcholesterol-homoarginine-methyl ester methylphosphonoamidate (PH55938;16).

FIG. 5 depicts a scheme for the synthesis ofcholesterol-lysine-amide-methylphosphonoamidate (PH55939; 17).

FIG. 6 depicts a scheme for the synthesis ofcholesterol-TREN-methylphosphonoamidate (PH55941; 18) andcholesterol-TREN-bis-guanidinium methylphosphonoamidate (PH55942; 19).

FIG. 7 depicts a scheme for the synthesis ofcholesterol-histidine-methylphosphonoamidate (PH55943; 20).

FIG. 8 depicts a scheme for the synthesis ofcholesterol-aminopropylimidazole-methylphosphonoamidate (PH55945; 21).

FIG. 9 depicts a scheme for the synthesis of vitamin-B6 andbeta-alanine-based cationic lipids.

FIG. 10 depicts a scheme for the synthesis of 2′-aminouridine-basedcationic lipids.

FIG. 11 depicts a scheme for the synthesis of vitamin-B6-cholesterolconjugate.

FIGS. 12A-G shows the secondary structure model for seven differentclasses of enzymatic nucleic acid molecules. Arrow indicates the site ofcleavage. --------- indicate the target sequence. Lines interspersedwith dots are meant to indicate tertiary interactions. - is meant toindicate base-paired interaction. FIG. 12A shows a Group I Intron motif:P1-P9.0 represent various stem-loop structures (Cech et al., 1994,Nature Struc. Bio., 1, 273). FIG. 12B shows an RNase P (M1RNA) motif:EGS represents external guide sequence (Forster et al., 1990, Science,249, 783; Pace et al., 1990, J. Biol. Chem., 265, 3587). FIG. 12C showsa Group II Intron motif: 5′SS means 5′ splice site; 3′SS means 3′-splicesite; IBS means intron binding site; EBS means exon binding site (Pyleet al., 1994, Biochemistry, 33, 2716). FIG. 12D shows a VS RNA motif:I-VI are meant to indicate six stem-loop structures; shaded regions aremeant to indicate tertiary interaction (Collins, International PCTPublication No. WO 96/19577). FIG. 12E shows a HDV Ribozyme motif: I-IVare meant to indicate four stem-loop structures (Been et al., U.S. Pat.No. 5,625,047). FIG. 12F shows a Hammerhead Ribozyme motif: I-III aremeant to indicate three stem-loop structures; stems I-III can be of anylength and may be symmetrical or asymmetrical (Usman et al., 1996, Curr.Op. Struct. Bio., 1, 527). FIG. 12G shows a Hairpin Ribozyme motif:H1-H4 are meant to indicate helices 1-4; Helix 1 and 4 can be of anylength; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; Bis guanosine, cytidine or uridine; V is adenosine, guanosine, orcytidine (Burke et al., 1996, Nucleic Acids & Mol. Biol., 10, 129;Chowrira et al., U.S. Pat. No. 5,631,359).

FIG. 13 depicts the structure of fluorescein-conjugated ribozyme and itssubstrate mRNA sequence. The fluorescein moiety (Fluor), attachedthrough an amino linker, does not reduce the enzymatic activity of theribozyme.

FIG. 14 depicts the chemical synthesis ofN²,N³-di-oleyl-(N,N′-diguanidinoethyl-aminoethane)-2,3-diaminopropionicacid.

FIG. 15 depicts the results of a blood clearance study using theEPC:CHOL:DOTAP:DSPE₂₀₀₀ liposome.

FIG. 16 depicts the results of a cellular inhibition study of IMPDH-2mRNA expression in Jurkat cells treated for 24 hours with IMPDHantisense and lipid NC 266.

FIG. 17 depicts the results of a cellular inhibition study of IMPDH-2mRNA expression in Jurkat cells treated for 24 hours with IMPDHantisense and lipid NC 267.

FIG. 18 depicts the results of a cellular inhibition study of IMPDH-2mRNA expression in Jurkat cells treated for 24 hours with IMPDHantisense and lipid NC 388.

Cationic lipids are bifunctional reagents (cationic head groupconjugated to a lipid tail) that include a positively charged group thatcan ion-pair with an anionic group present in a negatively chargedpolymer, such as a phosphate group present in a nucleic acidphosphodiester linkage or a phosphorothioate group present in nucleicacid having a modified phosphodiester linkage. In one embodiment, thecationic group ion-pairs with a negatively charged polymer to form alipid:polymer complex, such as a complex with a polynucleotide or apolypeptide (e.g. RNA, DNA, and protein). In another embodiment, thecationic group ion-pairs with RNA having enzymatic activity, such as aribozyme. Formation of the ion-pair increases the intrinsichydrophobicity of the negatively charged polymer and facilitatesdiffusion of the polymer across a cell membrane into the cell. Thelipid:polymer complex can contain more than one lipid molecule. In oneembodiment, a lipid:polymer complex contains lipid in an amount toion-pair with at least 50% of the anionic groups of a negatively chargedpolymer. In another embodiment, a lipid:polymer complex contains lipidin an amount to ion-pair with at least 90% of the anionic groups of anegatively charged polymer. The lipid of an lipid:polymer complex can bethe same or different. For example, the complex can contain lipidsdiffering in the cationic groups. The amount of cationic lipid andnegatively charged polymer which are combined to achieve the desiredamount of ionic pairing depends on the environment in which the lipidand the polymer is mixed, the type of lipid, and the type of polymer.The degree of ionic pairing can be measured by techniques known in theart (see, for example, U.S. Pat. No. 5,583,020, the contents of whichare incorporated by reference herein). In another embodiment, the lipidmolecule of the invention is provided in an amount of at least two toten times per negative charge on the polymer molecule.

Cationic lipids represent a subset of compounds in the broader class ofmulti-domain cellular transport vehicles (MCTVs). The MCTV family of theinvention includes single compounds as well as multi-component deliverysystems that incorporate structural domains designed to improve membranepermeability, cellular targeting, while reducing the nonspecificinteractions and toxicity of the incoming compound. In addition tocationic lipids, examples of MCTVs include transporters such as facialamphiphiles and other amphipathic compounds, carriers with targetingelements such as glycated moieties, peptides and vitamins, and liposomeswith fusogenic elements, pegylated lipids, and/or pH-sensitivecomponents.

The term “nucleic acid molecule” as used herein refers to a moleculecomprising nucleotides. The nucleic acid can be single, double, ormultiple stranded and can comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof. Nucleicacid molecule, for example, oligonucleotides, enzymatic nucleic acidmolecules, ribozymes, DNAzymes, antisense oligonucleotides, 2-5antisence chimera, triplex forming oligonucleotides, aptamers and othermolecules described herein, templates, primers, nucleic acid sensormolecules, reporter and or signal molecules.

The term “negatively charged molecules” as used herein, includesmolecules such as naturally occurring and chemically modified nucleicacid molecules (e.g., RNA, DNA, oligonucleotides, mixed polymers,peptide nucleic acid, and the like), peptides (e.g., polyaminoacids,polypeptides, proteins and the like), nucleotides, pharmaceutical andbiological compositions, that have negatively charged groups that canion-pair with the positively charged head group of the lipids of theinvention.

The term “compounds with neutral charge” as used herein refers tocompositions which are neutral or uncharged at neutral or physiologicalpH. Examples of such compounds include cholesterol (i.e., a steroidalalcohol, as defined in Lehninger, Biochemistry, 1982 ed., Worth Pub., p.315) and other steroids, cholesteryl hemisuccinate (CHEMS), dioleoylphosphatidyl choline, distearoylphosphotidyl choline (DSPC), fatty acidssuch as oleic acid, phosphatidic acid and its derivatives, phosphatidylserine, polyethylene glycol-conjugated phosphatidylamine,phosphatidylcholine, phosphatidylethanolamine and related variants,prenylated compounds including farnesol, polyprenols, tocopherol, andtheir modified forms, diacylsuccinyl glycerols, fusogenic or poreforming peptides, dioleoylphosphotidylethanolamine (DOPE), ceramide andthe like.

The term “head group” as used herein refers to an amino-containingmoiety that is positively charged and is capable of forming ion pairswith negatively charged regions of biopolymers such as nucleic acidmolecules.

The term “lipophilic group” as used herein refers to a hydrophobiclipid-containing group that facilitates transmembrane transport of thecationic lipid.

The term “linker” as used herein refers to a 1-6 atom carbon chain thatlinks the head group with the lipophylic group.

The term “ion pair” as used herein refers to a non-covalent interactionbetween oppositely charged groups.

The term “alkyl” as used herein refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain, and cyclic alkylgroups. In one embodiment, the alkyl group has 1 to 12 carbons. Inanother embodiment the alkyl is a lower alkyl of from 1 to 7 carbons,for example 1 to 4 carbons. The alkyl group can be substituted orunsubstituted. When substituted the substituted group(s) can be forexample a, hydroxy, cyano, alkoxy, NO₂ or N(CH₃)₂, amino, or SH group.

The term “alkoxy” as used herein refers to an OR group, wherein R is analkyl.

The terms “aryl” as used herein refers to an aromatic group which has atleast one ring having a conjugated π electron system and includescarbocyclic aryl, heterocyclic aryl and biaryl groups, all of which canbe optionally substituted. Non-limiting examples of substituent(s) thatcan exist on aryl groups are halogen, trihalomethyl, hydroxyl, SH,cyano, alkoxy, alkyl, alkenyl, alkynyl, and/or amino groups.

The term “alkenyl” as used herein refers to unsaturated hydrocarbongroups containing at least one carbon-carbon double bond, includingstraight-chain, branched-chain, and cyclic groups. In one embodiment,the alkenyl group has 1 to 12 carbons. In another embodiment, thealkenyl group is a lower alkenyl of from 1 to 7 carbons, for example 1to 4 carbons. The alkenyl group can be substituted or unsubstituted.When substituted the substituted group(s) can include, hydroxyl, cyano,alkoxy, NO₂, halogen, N(CH₃)₂, amino, and/or SH groups.

The term “alkynyl” as used herein refers to an unsaturated hydrocarbongroup containing at least one carbon-carbon triple bond, includingstraight-chain, branched-chain, and cyclic groups. In one embodiment,the alkynyl group has 1 to 12 carbons. In another embodiment the alkynylgroup is a lower alkynyl of from 1 to 7 carbons, for example 1 to 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) can include, hydroxyl, cyano,alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino and/or SH groups.

The term “alkylaryl” as used herein refers to an alkyl group (asdescribed above) covalently joined to an aryl group (as describedabove).

The terms “carbocyclic aryl” as used herein refers to groups wherein thering atoms on the aromatic ring are all carbon atoms. The carbon atomsare optionally substituted.

The terms “heterocyclic aryl” as used herein refers to groups havingfrom 1 to 3 heteroatoms as ring atoms in the aromatic ring and theremainder of the ring atoms are carbon atoms. Suitable heteroatomsinclude oxygen, sulfur, and nitrogen, and include furanyl, thienyl,pyridyl, pyrrolyl, pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and thelike, all optionally substituted.

The term “acyl” as used herein refers to —C(O)R groups, wherein R is analkyl or aryl.

The term “lipid aggregate” as used herein refers to a lipid-containingcomposition (i.e., a composition comprising a lipid according to theinvention) wherein the lipid is in the form of a liposome, micelle(non-lamellar phase) or other aggregates with one or more lipids.

The term “lipid formation” as used herein refers to a formulationcomprising at least one lipid molecule, such as those described herein,and at least one molecule to be delivered, for example, a nucleic acid,polynucleotide, oligonucleotide, peptide, polypeptide, protein,carbohydrate, steroid, polymer, metal, or small molecule.

The term “suspension cells” as used herein refers to cells that do notrequire the attachment to a solid substrate (e.g. plastic surface)(anchorage dependence), in order to survive or proliferate. Non-limitingexamples of these cells include hemopoietic, transformed, or cancercells.

The term “T cell” as used herein refers to cells responsible for cellmediated immunity which originate in the thymus gland (e.g. jurkatcells).

The terms “oligonucleotide” or “polynucleotide” as used herein refers toa nucleic acid molecule comprising a stretch of three or morenucleotides.

The term “enzymatic nucleic acid molecule” as used herein refers to anucleic acid molecule which has complementarity in a substrate bindingregion to a specified gene target, and also has an enzymatic activitywhich is active to specifically cleave target RNA. That is, theenzymatic nucleic acid molecule is able to intermolecularly cleave RNAand thereby inactivate a target RNA molecule. These complementaryregions allow sufficient hybridization of the enzymatic nucleic acidmolecule to the target RNA and thus permit cleavage. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% canalso be useful in this invention (see for example Werner and Uhlenbeck,1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids canbe modified at the base, sugar, and/or phosphate groups. The termenzymatic nucleic acid is used interchangeably with phrases such asribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme oraptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The specific enzymatic nucleic acid molecules described in the instantapplication are not limiting in the invention and those skilled in theart will recognize that all that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target nucleicacid regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart a nucleic acidcleaving and/or ligation activity to the molecule (Cech et al., U.S.Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).

Several varieties of enzymatic nucleic acids are known presently, whichcan catalyze, for example, the hydrolysis of RNA phosphodiester bonds intrans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids with RNA endonucleaseactivity act by first binding to a target RNA. Such binding occursthrough the target binding portion of a enzymatic nucleic acid which isheld in close proximity to an enzymatic portion of the molecule thatacts to cleave the target RNA. Thus, the enzymatic nucleic acid, forexample, first recognizes and then binds a target RNA throughcomplementary base-pairing, and once bound to the correct site, actsenzymatically to cut the target RNA. Strategic cleavage of such a targetRNA will destroy its ability to direct synthesis of an encoded protein.After an enzymatic nucleic acid has bound and cleaved its RNA target, itis released from that RNA to search for another target and canrepeatedly bind and cleave new targets. Thus, a single ribozyme moleculeis able to cleave many molecules of target RNA. In addition, theenzymatic nucleic acid is a highly specific inhibitor of geneexpression, with the specificity of inhibition depending not only on thebase-pairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of an enzymatic nucleic acid molecule.

The term “sufficient length” as used herein refers to an oligonucleotideof greater than or equal to 3 nucleotides that is of a length greatenough to provide the intended function under the expected condition.For example, for binding arms of enzymatic nucleic acid “sufficientlength” means that the binding arm sequence is long enough to providestable binding to a target site under the expected binding conditions.Preferably, the binding arms are not so long as to prevent usefulturnover of the nucleic acid molecule.

The term “stably interact” as used herein refers to interaction of theoligonucleotides of the invention with a target nucleic acid (e.g., byforming hydrogen bonds with complementary nucleotides in the targetunder physiological conditions) that is sufficient for an intendedpurpose (e.g., cleavage of target RNA by an enzyme).

By “antisense nucleic acid”, as used herein refers to a non-enzymaticnucleic acid molecule that binds to target nucleic acid by means ofRNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993Nature 365, 566) interactions and alters the activity of the targetnucleic acid (for a review, see Stein and Cheng, 1993 Science 261, 1004and Woolf et al., U.S. Pat. No. 5,849,902). Typically, antisensemolecules are complementary to a target sequence along a singlecontiguous sequence of the antisense molecule. However, in certainembodiments, an antisense molecule can bind to substrate such that thesubstrate molecule forms a loop, and/or an antisense molecule can bindsuch that the antisense molecule forms a loop. Thus, the antisensemolecule can be complementary to two (or even more) non-contiguoussubstrate sequences or two (or even more) non-contiguous sequenceportions of an antisense molecule can be complementary to a targetsequence or both. For a review of current antisense strategies, seeSchmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al.,1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. DrugDev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998,Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol.,40, 1-49. In addition, antisense DNA can be used to target nucleic acidby means of DNA-RNA interactions, thereby activating RNase H, whichdigests the target nucleic acid in the duplex. The antisenseoligonucleotides can comprise one or more RNAse H activating region,which is capable of activating RNAse H cleavage of a target nucleicacid. Antisense DNA can be synthesized chemically or expressed via theuse of a single stranded DNA expression vector or equivalent thereof.

By “RNase H activating region” as used herein refers to a region(generally greater than or equal to 4-25 nucleotides in length,preferably from 5-11 nucleotides in length) of a nucleic acid moleculecapable of binding to a target nucleic acid to form a non-covalentcomplex that is recognized by cellular RNase H enzyme (see for exampleArrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No.5,989,912). The RNase H enzyme binds to the nucleic acid molecule-targetnucleic acid complex and cleaves the target nucleic acid sequence. TheRNase H activating region comprises, for example, phosphodiester,phosphorothioate (preferably at least four of the nucleotides arephosphorothiote substitutions; more specifically, 4-11 of thenucleotides are phosphorothiote substitutions); phosphorodithioate,5′-thiophosphate, or methylphosphonate backbone chemistry or acombination thereof. In addition to one or more backbone chemistriesdescribed above, the RNase H activating region can also comprise avariety of sugar chemistries. For example, the RNase H activating regioncan comprise deoxyribose, arabino, fluoroarabino or a combinationthereof, nucleotide sugar chemistry. Those skilled in the art willrecognize that the foregoing are non-limiting examples and that anycombination of phosphate, sugar and base chemistry of a nucleic acidthat supports the activity of RNase H enzyme is within the scope of thedefinition of the RNase H activating region and the instant invention.

By “2-5A antisense chimera” as used herein refers to an antisenseoligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylateresidue. These chimeras bind to target nucleic acid in asequence-specific manner and activate a cellular 2-5A-dependentribonuclease which, in turn, cleaves the target nucleic acid (Torrenceet al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al.,2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998,Pharmacol. Ther., 78, 55-113).

By “triplex forming oligonucleotides” as used herein refers to anoligonucleotide that can bind to a double-stranded polynucleotide, suchas DNA, in a sequence-specific manner to form a triple-strand helix.Formation of such triple helix structure has been shown to inhibittranscription of the targeted gene (Duval-Valentin et al., 1992 Proc.Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37;Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181-206).

By “nucleic acid decoy molecule”, or “decoy” as used herein refers to anucleic acid molecule that mimics the natural binding domain for aligand. The decoy therefore competes with the natural binding target forthe binding of a specific ligand. For example, it has been shown thatover-expression of HIV trans-activation response (TAR) RNA can act as a“decoy” and efficiently binds HIV tat protein, thereby preventing itfrom binding to TAR sequences encoded in the HIV RNA (Sullenger et al.,1990, Cell, 63, 601-608).

By “aptamer” or “nucleic acid aptamer” as used herein refers to anucleic acid molecule that binds specifically to a target moleculewherein the nucleic acid molecule has sequence that is distinct fromsequence recognized by the target molecule in its natural setting.Alternately, an aptamer can be a nucleic acid molecule that binds to atarget molecule where the target molecule does not naturally bind to anucleic acid. The target molecule can be any molecule of interest. Forexample, the aptamer can be used to bind to a ligand binding domain of aprotein, thereby preventing interaction of the naturally occurringligand with the protein, see for example Gold et al., U.S. Pat. Nos.5,475,096 and 5,270,163; Gold et al., 1995, Annu. Rev. Biochem., 64,763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin.Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann andPatel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry,45, 1628.

The term “double stranded RNA” or “dsRNA” as used herein refers to adouble stranded RNA molecule capable of RNA interference “RNAi”,including short interfering RNA “siRNA” see for example Bass, 2001,Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; andKreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914.

The term “nucleotide” as used herein refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are recognized in the art to include natural bases(standard), and modified bases well known in the art. Such bases aregenerally located at the 1′ position of a nucleotide sugar moiety.Nucleotides generally comprise a base, sugar and a phosphate group. Thenucleotides can be unmodified or modified at the sugar, phosphate and/orbase moiety, (also referred to interchangeably as nucleotide analogs,modified nucleotides, non-natural nucleotides, non-standard nucleotidesand other; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; Uhlman & Peyman, supraall are hereby incorporated by reference herein). There are severalexamples of modified nucleic acid bases known in the art as summarizedby Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of chemically modified and other natural nucleicacid bases that can be introduced into nucleic acids include, inosine,purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra).

By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents; such bases can be used at any position, for example, withinthe catalytic core or substrate binding regions of the enzymatic nucleicacid domain of an nucleic acid sensor molecule. Such modified bases canalso be present at one or more positions within the sensor domain of thenucleic acid sensor molecule, for example to improve interaction withthe target nucleic acid sequence.

The term “unmodified nucleotide” as used herein refers to a nucleotidewith one of the bases adenine, cytosine, guanine, thymine, uracil joinedto the 1′ carbon of beta-D-ribo-furanose.

The term “modified nucleotide” as used herein refers to a nucleotidethat contains a modification in the chemical structure of an unmodifiednucleotide base, sugar and/or phosphate.

By “Inozyme” or “NCH” motif or configuration is meant, an enzymaticnucleic acid molecule comprising a motif as is generally described asNCH Rz in Ludwig et al., International PCT Publication No. WO 98/58058and U.S. patent application Ser. No. 08/878,640. Inozymes possessendonuclease activity to cleave RNA substrates having a cleavage tripletNCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridineor cytidine, and/represents the cleavage site. Inozymes can also possessendonuclease activity to cleave RNA substrates having a cleavage tripletNCN/, where N is a nucleotide, C is cytidine, and/represents thecleavage site

By “G-cleaver” motif or configuration is meant, an enzymatic nucleicacid molecule comprising a motif as is generally described in Ecksteinet al., U.S. Pat. No. 6,127,173 and in Kore et al., 1998, Nucleic AcidsResearch 26, 4116-4120. G-cleavers possess endonuclease activity tocleave RNA substrates having a cleavage triplet NYN/, where N is anucleotide, Y is uridine or cytidine and/represents the cleavage site.G-cleavers can be chemically modified.

By “amberzyme” motif or configuration is meant, an enzymatic nucleicacid molecule comprising a motif as is generally described in Beigelmanet al., International PCT publication No. WO 99/55857 and U.S. patentapplication Ser. No. 09/476,387. Amberzymes possess endonucleaseactivity to cleave RNA substrates having a cleavage triplet NG/N, whereN is a nucleotide, G is guanosine, and/represents the cleavage site.Amberzymes can be chemically modified to increase nuclease stability. Inaddition, differing nucleoside and/or non-nucleoside linkers can be usedto substitute the 5′-gaaa-3′ loops of the motif. Amberzymes represent anon-limiting example of an enzymatic nucleic acid molecule that does notrequire a ribonucleotide (2′-OH) group within its own nucleic acidsequence for activity.

By “zinzyme” motif or configuration is meant, an enzymatic nucleic acidmolecule comprising a motif as is generally described in Beigelman etal., International PCT publication No. WO 99/55857 and U.S. patentapplication Ser. No. 09/918,728. Zinzymes possess endonuclease activityto cleave RNA substrates having a cleavage triplet including but notlimited to, YG/Y, where Y is uridine or cytidine, and G is guanosineand/represents the cleavage site. Zinzymes can be chemically modified toincrease nuclease stability through various substitutions, includingsubstituting 2′-O-methyl guanosine nucleotides for guanosinenucleotides. In addition, differing nucleotide and/or non-nucleotidelinkers can be used to substitute the 5′-gaaa-2′ loop of the motif.Zinzymes represent a non-limiting example of an enzymatic nucleic acidmolecule that does not require a ribonucleotide (2′-OH) group within itsown nucleic acid sequence for activity.

The term “DNAzyme” as used herein refers to an enzymatic nucleic acidmolecule that does not require the presence of a 2′-OH group within itfor its activity. In particular embodiments the enzymatic nucleic acidmolecule can have an attached linker(s) or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. DNAzymes can be synthesized chemically or expressedendogenously in vivo, by means of a single stranded DNA vector orequivalent thereof. Examples of DNAzymes are generally reviewed in Usmanet al., International PCT Publication No. WO 95/11304; Chartrand et al.,1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro etal., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17,422-423; and Santoro et. al., 2000, J. Am. Chem. Soc., 122, 2433-39;Perrin et al., 2001, JACS., 123, 1556. Additional DNAzyme motifs can beselected for using techniques similar to those described in thesereferences, and hence, are within the scope of the present invention.

The term “RNA” as used herein refers to a molecule comprising at leastone ribonucleotide residue. By “ribonucleotide” or “2′-OH” is meant anucleotide with a hydroxyl group at the 2′ position of aβ-D-ribo-furanose moiety.

The term “system” or “biological system” as used herein refers to agroup of substances or components that can be collectively combined oridentified. A system can comprise a biological system, for example, anorganism, cell, or components, extracts, and samples thereof. A systemcan further comprise an experimental or artificial system, where varioussubstances or components are intentionally combined together. The“biological system” as used herein can be a eukaryotic system or aprokaryotic system, for example, a bacterial cell, plant cell or amammalian cell, or of plant origin, mammalian origin, yeast origin,Drosophila origin, or archebacterial origin.

The term “cation” as used herein refers to a positively chargedmolecule.

The term “vitamin” as used herein refers to a small molecule, such asriboflavin, nicotinamide, biotin, thiamine, lipoic acid, retinal,pyridoxal, folate, pantothenic acid, cyanocobalamin, aminopterin, andtheir respective analogs, which bind to a specific protein andparticipate directly in enzyme catalysis.

Method of Use

The cationic lipid molecules of the instant invention can be used toadminister negatively charged polymers which act as pharmaceuticalagents. Pharmaceutical agents prevent, inhibit the occurrence, or treat(alleviate a symptom to some extent, preferably all of the symptoms) ofa disease state in a patient.

The term “patient” as used herein refers to an organism which is a donoror recipient of explanted cells or the cells themselves. “Patient” alsorefers to an organism to which the compounds of the invention can beadministered. Preferably, a patient is a mammal, e.g., a human, primateor a rodent.

Generally, these molecules are used in solution with the negativelycharged polymer to be administered (e.g., RNA, DNA or protein) andintroduced by any standard means, with or without stabilizers, buffers,and the like, to form a pharmaceutical composition. When it is desiredto use a liposome delivery mechanism, standard protocols for formationof liposomes can be followed. The compositions of the present inventionmay also be formulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions; suspensions for injectable administration; and the like.

The present invention also includes pharmaceutically acceptableformulations of the compounds described above, preferably in combinationwith the negatively charged polymer to be delivered. These formulationsinclude salts of the above compounds, e.g., acid addition salts, forexample, salts of hydrochloric, hydrobromic, acetic acid, and benzenesulfonic acid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or patient, preferably a human. Suitableforms, in part, depend upon the use or the route of entry, for exampleoral, transdermal, or by injection. Such forms should not prevent thecomposition or formulation to reach a target cell (i.e., a cell to whichthe negatively charged polymer is desired to be delivered to). Forexample, pharmacological compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms which prevent the compositionor formulation from exerting its effect.

The term “systemic administration” as used herein refers to in vivosystemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes which lead to systemic absorption include, without limitations:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexpose the desired negatively charged polymers, e.g., nucleic acids, toan accessible diseased tissue. The rate of entry of a drug into thecirculation has been shown to be a function of molecular weight or size.The use of a liposome or other drug carrier comprising the compounds ofthe instant invention can potentially localize the drug, for example, incertain tissue types, such as the tissues of the reticular endothelialsystem (RES). A liposome formulation which can facilitate theassociation of drug with the surface of cells, such as, lymphocytes andmacrophages is also useful. This approach may provide enhanced deliveryof the drug to target cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of abnormal cells, such asthe cancer cells.

In one embodiment, the invention features the use of the cationic lipidsof the invention in a composition comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes). These formulationsoffer an method for increasing the accumulation of drugs in targettissues. This class of drug carriers resists opsonization andelimination by the mononuclear phagocytic system (MPS or RES), therebyenabling longer blood circulation times and enhanced tissue exposure forthe encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627;Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomeshave been shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; Choi et al., International PCT Publication No. WO 96/10391;Ansell et al., International PCT Publication No. WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392; all of these areincorporated by reference herein). Long-circulating liposomes are alsolikely to protect drugs from nuclease degradation to a greater extentcompared to cationic liposomes, based on their ability to avoidaccumulation in metabolically aggressive MPS tissues such as the liverand spleen. All of these references are incorporated by referenceherein.

The present invention also includes compositions prepared for storage oradministration which include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985)hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents may be provided. Id. at 1449.These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentsmay be used. Id.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors which those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The molecules of the invention and formulations thereof can beadministered orally, topically, parenterally, by inhalation or spray orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a molecule of theinvention and a pharmaceutically acceptable carrier. One or more nucleicacid molecules of the invention can be present in association with oneor more non-toxic pharmaceutically acceptable carriers and/or diluentsand/or adjuvants, and if desired other active ingredients. Thepharmaceutical compositions containing molecules of the invention can bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The molecules of the invention can also be administered in the form ofsuppositories, e.g., for rectal administration of a compound. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Molecules of the invention can be administered parenterally in a sterilemedium. A drug, depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anesthetics, preservatives and buffering agentscan be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular patientdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The molecules of the present invention can also be administered to apatient in combination with other therapeutic compounds to increase theoverall therapeutic effect. The use of multiple compounds to treat anindication can increase the beneficial effects while reducing thepresence of side effects.

The examples provided herein illustrate different aspects andembodiments of the present invention. Although the examples presentedhere primarily pertain to delivery of ribozymes and plasmid DNA, oneskilled in the art will recognize that any nucleic acid, protein, lipid,or another molecule, either alone or in combinations can be delivered totarget biological system using the teachings of the present invention.These examples are not intended in any way to limit the disclosedinvention.

Example 1 Synthesis of Diaminobutyric Acid and Guanidinium-BasedCationic Lipids (FIG. 2)

Synthesis of palmityloleylamine (1): 1-bromohexadecane (15.27 g, 50mmol) was rapidly added to oleylamine (26.75 g, 100 mmol) at 100° C. Thereaction mixture was heated at 120° C. for 30 minutes and than cooled toroom temperature. Chloroform (200 ml) was added followed by 1 N NaOH (50ml). The mixture was then extracted with H₂O (200 ml), the organic layerdried (Na₂SO₄) and concentrated to a syrup. Silica gel columnchromatography using 5-20% gradient methanol in dichloromethane afforded20.5 g of palmityloleylamine as a syrup (yield, 83%). The identity ofthe product was confirmed using NMR spectroscopy. ¹H NMR (CDCl₃) d 5.34(m, 2H, CH═CH), 2.58 (m, 4H), 2.00 (m, 4H), 1.47 (m, 4H), 1.25 (m, 48H),0.86 (m, 6H). FAB-MS: 493 [M+H]⁺. (Other reagents could includeoleyl-bromide and hexadecane amine)

Synthesis of N′-palmityl-N′-oleyl-N-CBZ-glycinamide (2): (1) (2.46 g, 5mmol) was added to a solution of N-CBZ-glycine N-hydroxysuccinimideester (3.06 g, 10 mmol) suspended in dichloromethane (1.39 ml)containing triethylamine (TEA) (10 mmol). The reaction mixture wasstirred at room temperature overnight and then concentrated to an oilunder vacuum. Silica gel chromatography using 1-5% gradient methanol indichloromethane gave 1.54 g of N′-palmityl-N′-oleyl-N-CBZ-glycinamide(yield, 45%). ¹H NMR (CDCl₃) d 7.35 (m, phenyl), 5.83 (br s, NH), 5.35(m, CH═CH), 5.12 (s, 2H, CH₂Ph), 4.00 (m, 2H, glycyl), 3.31 (m, 2H),3.13 (m, 2H), 2.00 (m, 4H), 1.53 (m, 4H), 1.25 (m, 48H), 0.88 (m, 6H).

Synthesis of N′-palmityl-N′-oleyl-glycinamide (3): 10% Palladium onCarbon (Pd/C) was added to N′-palmityl-N′-oleyl-N-CBZ-glycinamide (0.5g, 0.73 mmol) dissolved in absolute ethanol (3 ml) under argon gas. Theflask was immersed in a 20° C. water bath prior to the addition of1,4-cyclohexadiene (0.66 ml). The reaction mixture was stirred at roomtemperature overnight, the catalyst was filtered off and the filtrateevaporated to dryness giving 0.3 g of product (yield, 75%). ¹H NMR(acetone-d₆) d 5.41 (m, 2H, CH═CH), 4.07 (br s, 2H, glycyl), 3.36 (m,2H), 3.29 (m, 2H), 2.80 (br s, NH₂), 2.05 (m, 2H), 1.98 (m, 2H), 1.63(m, 2H), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS: 549 [M+H]⁺.

Synthesis ofN′-palmityl-N′-oleyl-alpha,gamma-bis-Boc-diaminobutyryl-glycinamide (4):The mixture of 3 (1.12 g, 2.04 mmol),N-alpha-N-gamma-di-Boc-diaminobutyric acid (631 mg, 2.24 mmol),2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (553 mg, 2.24mmol) in CH₂Cl₂ was stirred for 1 hour at room temperature. The mixturewas then concentrated to a syrup and 1.2 g of the product was isolatedby column chromatography using 20-50% gradient of hexanes in ethylacetate (yield, 69%). ¹H NMR (CDCl₃) d 7.14 (br s, NH), 5.38 (m, 2H,CH═CH), 5.28 (br s, 1H, NH), 5.12 (br s, 1H, NH), 4.02 (m, 2H, glycyl),3.42 (m, 1H), 3.31 (m, 2H), 3.15 (m, 2H), 3.02 (m, 2H), 1.95 (m, 4H),1.77 (m, 2H), 1.53 (m, 4H), 1.25 (m, 48H). FAB-MS: 850 [M+H]⁺.

Synthesis of N′-palmityl-N′-oleyl-alpha,gamma-diaminobutyryl-glycinamide (JA59311) (5): compound 4 (350 mg, 0.41mmol) was dissolved in dioxane (6 ml) followed by the addition of 4 MHCl in dioxane (6 ml). The reaction mixture was stirred at roomtemperature for 2 hours, than concentrated in vacuo and azeotroped twicewith toluene. The residue was partitioned between CH₂Cl₂ and 0.2 N NaOH,the organic layer was washed with saturated NaHCO₃ solution, and thendried (Na₂SO₄) and evaporated to dryness. Flash silica gelchromatography using CH₂Cl₂/methanol/conc. NH₄OH 40:10:2 yielded 200 mgof compound 5 (yield, 75%). ¹H NMR (CDCl₃) d 8.06 (br s, 1H, NH), 5.38(m, 2H, CH═CH), 4.04 (m, 2H, glycyl), 3.54 (m, 1H), 3.31 (m, 2H), 3.17(m, 2H), 2.86 (m, 2H), 1.93 (m, 4H), 1.67 (m, 2H), 1.54 (m, 4H), 1.41(br s, 4H, NH₂), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS: 650 [M+H]⁺.

Synthesis ofN′-palmityl-N′-oleyl-N-gamma-carboxamidine-alpha,gamma-diaminobutyryl-glycinamide(JA59312) (6): To the solution of 5 (0.16 g, 0.25 mmol) anddiisopropylethylamine (DIPEA) (83 mL) in THF/methanol 1:1 (0.8 ml),1H-pyrazole-1-carboxamidine hydrochloride (70 mg, 0.48 mmol) was addedunder argon gas. The reaction mixture was stirred at room temperatureovernight and then concentrated to a syrup. Silica gel columnchromatography using CH₂Cl₂ followed by CH₂Cl₂/methanol/conc. NH₄OH40:10:2 yielded 50 mg of compound 6 (yield, 29%). ¹H NMR (CDCl₃) d 5.37(m, 2H, CH═CH), 4.07 (m, 2H, glycyl), 3.94 (m, 1H), 3.42 (m, 2H), 3.21(m, 4H), 2.01 (m, 6H), 1.58 (m, 4H), 1.46 (m, 2H, NH₂), 1.25 (m, 48H),0.87 (m, 6H). FAB-MS: 692 [M+H]⁺.

Synthesis ofN′-palmityl-N′-oleyl-alpha,gamma-bis-trimethylammoniumbutyryl-glycinamide(JA59316) (7): A dihydrogenchloride salt of 5 (130 mg, 0.2 mmol) wasdissolved in methanol (4 ml) and combined with KHCO₃ (0.2 g) and CH₃I(0.2 ml). The mixture was then stirred at room temperature for 3 days.The reaction mixture was then filtered through the bed of Celite,followed by filtration through 0.45 m PTFE filter. The filtrate was thenevaporated to dryness affording 160 mg of the desired product (yield,94%). ¹H NMR (CDCl₃) d 3.58 [s, 9H, (CH₃)₃], 3.44 [s, 9H, (CH₃)₃].

Synthesis of N′-palmityl-N′-oleyl-N-carboxamidine-glycinamide.HCl(JA59314) (8): Using the same procedure described above for thepreparation of 6 except that crystallization from methanol instead ofcolumn chromatography was used for purification, 8 was prepared in 51%yield. ¹H NMR (CDCl₃) d 7.70-7.25 (m, 5H, NH), 5.38 (m, 2H, CH═CH), 4.25(m, 2H, glycyl), 3.27 (m, 4H), 1.96 (m, 4H), 1.53 (m, 4H), 1.25 (m,48H), 0.87 (m, 6H). FAB-MS: 592 [M+H]⁺.

Synthesis of N′-palmityl-N′-oleyl-guanidine (JA59317) (9): The mixtureof a hydrochloride salt of 1 (285 mg, 0.54 mmol), cyanamide (50 mg, 1.19mmol) and 1-butanol (2 ml) was stirred at 120° C. for 2 hours. Thecooled mixture was diluted with CH₂Cl_(2 (50 ml)) and washed withsaturated aqueous NaCl(Brine)/methanol 1:1 (50 ml). The organic layerwas then dried (Na₂SO₄), evaporated to an oil and chromatographed on acolumn of silica gel using CH₂Cl₂/methanol/conc. NH₄OH 40:10:2 giving 80mg of the desired material (yield, 28%). ¹H NMR (CDCl₃) d 7.08 (br s,1H, NH), 5.34 (m, 2H, CH═CH), 3.29 (m, 4H), 2.00 (m, 4H), 1.62 (m, 4H),1.25 (m, 48H), 0.88 (m, 6H). FAB-MS: 535 [M+H]⁺.

Example 2 Synthesis of DS 46596 (12)

Synthesis of Cholesterylamine (10): Referring to FIG. 3, cholesterylchloride (10 grams, 25 mmol) was partially dissolved in dry methanol (50ml) and the solution was heated with stirring to 155° C. for 18 hr at500 psig using a 300 ml Parr bomb apparatus charged with dry ammoniagas. The bomb was cooled to room temperature and the methanol wasremoved by steam distillation on a rotary evaporator. Compound 10 waspurified using E. Merck silica chromatography by eluting withdichloromethane/methanol (4:1 v/v) to yield 4 grams of the ninhydrinpositive product (yield, 60%). Identity was confirmed by ES-MS.

Synthesis of Boc₃arginineNHcholesterylamide (11): A 200 mL pear shapedflask with stir bar was charged with a mixture of 10 (1 g, 2.6 mmol),Boc₃ arginine (1.2 grams, 2.6 mmol), diisopropylcarbodiimide (450 ul,2.9 mmol) and dichloromethane (70 mls). The mixture of reagents wasstirred at room temperature for two hours. Following the reaction, thesolution was washed with aqueous sodium bicarbonate (5% w/v) and theorganic layer was separated and dried to a solid using a rotaryevaporator. Compound 11 was dissolved in 5 mls of dichloromethane priorto purification using silica gel chromatography. (yield, 90%). Identitywas confirmed by ES-MS. ¹H NMR (dmso-d6): . ¹H NMR (dmso-d6): 9.32 (bd),6.63 (d), 5.30 (m), 4.00 (m), 3.80 (m) 3.40 (m), 1.539 (s, tBoc), 1.517(s, tBoc), 1.497 (s, tBoc).

Synthesis of Boc arginineNHcholesterylamide (DS4659) (12): Compound 11(50 mg, 60 umol) was dissolved in anhydrous 1,4-dioxane (300 ul) andcombined with 4M HCl in dioxane (400 ul). The mixture was left at roomtemperature for 2 hours and the reaction was stopped by removing allsolvent and HCl using a stream of dry nitrogen gas. Compound 12 wasisolated using a wide pore C18 silica column and an isocraticmethanol:water (88:12) eluant with detection at 210 nm. Fractionationallowed recovery of 20 mgs of compound 12 (yield, 44%). Identity wasconfirmed by ES-MS. ¹H NMR (dmso-d6): 7.82 (d), 6.36 (bs), 3.51 (m),3.43 (m), 3.33 (m) 3.15 (m), 1.472 (s, tBoc).

Example 3 Synthesis of PH 55933 (15) and PH 55938 (16)

Synthesis of (13): Referring to the FIG. 4, to a solution ofmethylphosphonic dichloride (0.332 g, 2.5 mmol, ³¹P NMR s, 43.93 ppm)stirring at room temperature under positive pressure argon was added4-dimethylaminopyridine (DMAP) (0.31 g, 2.5 mmol). The resulting clear,colorless solution was cooled to −70° C. and a solution of cholesterol(0.97 g, 2.5 mmol) suspended in anhydrous dichloromethane (20 ml) wasadded via syringe with vigorous stirring over a period of one hour. Thereaction mixture was allowed to warm to room temperature and wasmaintained at room temperature for 18 hours at which time ³¹P NMRanalysis of a small aliquot of the reaction mixture indicated completereaction (d, 39.08 ppm).

Crude (13) was treated with additional DMAP (0.31 g, 2.5 mmol) and thereaction mixture cooled to −70° C. while stirring under positivepressure argon. H-Lys(Z)-OCH₃ (0.66 g, 2.25 mmol) in anhydrousdichloromethane (20 ml) was added dropwise via syringe over a period ofone hour. The reaction mixture was warmed to room temperature andstirred for an additional 18 hours (reaction complete by ³¹P NMR).Direct loading onto flash silica followed by a gradient of 0 to 10%EtOAc/hexanes then 5% EtOH/dichloromethane gave 1.12 g of (14) (Yield,60% over two steps). ³¹P NMR (s, 30.98 ppm).

Synthesis of Cholesterol-Lysine-methylphosphonoamidate (PH55933) (15):Compound (14) (1.0 g, 1.35 mmol) was dissolved in anhydrous EtOH andcooled to 0° C. with an ice/water bath while stirring under argon. 10Pd/c (1.0 g, 1 mass eq.) was added to the reaction mixture followed bydropwise addition of 1,4 cyclohexadiene (1.27 ml, 13.5 mmol). Afterwarming to room temperature, the reaction was complete after 4 hours asdetermined by TLC (15% MeOH/dichloromethane). The reaction mixture wasfiltered over celite and dried in vacuo. Flash chromatography utilizinga gradient of 5 to 15% MeOH/dichloromethane 1% TEA afforded 0.64 g of(15): (yield, 78%) ³¹P NMR (s, 30.88 ppm), mass spec. calcd=606.87,found=607.47.

Synthesis of Cholesterol-Homoarginine-methylphosphonoamidate (PH55938)(16): To a solution of (15) (0.131 g, 0.216 mmol) stirring at roomtemperature under argon in anhydrous DMF (2.0 ml),1-H-pyrazole-1-carboxamidine.HCl (32 mg, 0.216 mmol) was added followedby diisopropylethylamine (28 ml, 0.216 mmol). The reaction mixture wasstripped slightly on a rotovap then rotated overnight without vacuum atroom temperature. After removing DMF in vacuo the reaction residue wasdissolved in dichloromethane and applied to a flash silica gel column.An isocratic system of 20% MeOH/dichloromethane, 2% NH₄OH followed bytreatment with Dowex OH⁻ (300 mg) in MeOH gave 80 mg of desired product(yield, 57%). ³¹P NMR (s, 31.99 ppm), mass spec. calcd=648.91,found=649.48.

Example 4 Synthesis of PH 55939 (17)

Synthesis of Cholesterol-Lysine-methylphosphonoamidate amide (PH55939)(17): Referring to FIG. 5, compound (15) (76 mg, 0.125 mmol) was treatedwith a 0° C. saturated methanolic ammonia solution (5 ml) at roomtemperature for 18 hours (some venting required). The reaction mixturewas evaporated in vacuo then purified by flash silica gel chromatographyto give 45 mg of product (yield, 61%). ³¹P NMR (s, 32.17 ppm), massspec. calcd=591.86, found=592.23.

Example 5 Synthesis of PH 55941 (18) and PH 55942 (19)

Synthesis of Cholesterol-TREN-methylphosphonamidate (PH55941) (18):Referring to FIG. 6, 4-Dimethylaminopyridine (DMAP) (0.31 g, 2.5 mmol)was added to a solution of methylphosphonic dichloride (0.332 g, 2.5mmol, ³¹P NMR s, 43.93 ppm) stirring at room temperature under positivepressure argon gas. The resulting clear, colorless solution was cooledto −70° C. and a solution of cholesterol (0.97 g, 2.5 mmol) in anhydrousdichloromethane (20 ml) was added via syringe with vigorous stirringover a period of one hour. The reaction mixture was allowed to warm toroom temperature and was maintained for 18 hours at which time ³¹P NMRanalysis of a small aliquot of the reaction mixture indicated completereaction (d, 39.08 ppm). Crude (13) was treated with additional DMAP(0.31 g, 2.5 mmol) and the reaction mixture cooled to −70° C. whilestirring under positive pressure argon. Tris(2-aminoethyl)amine (TREN)(0.37 ml, 2.5 mmol) in anhydrous dichloromethane (20 ml) was addeddropwise via syringe over a period of two hours. The reaction mixturewas warmed to room temperature and stirred for an additional 18 hours(reaction complete by ³¹P NMR). Direct loading onto flash silicafollowed by a gradient of 10 to 20% MeOH/dichloromethane with 1 to 4%NH₄OH gave 0.442 g of (18) as a white foam: (yield, 28% over two steps),³¹P NMR (d, 32.57 ppm), mass spec. calcd=592.89, found=593.49.

Synthesis of Cholesterol-TREN-bis-guanidinium methylphosphonamidate(PH55942) (19): Compound (18) (0.148 g, 0.25 mmol) was dissolved inanhydrous DMF (1.0 ml) and anhydrous dichloromethane (5.0 ml).1-H-pyrazole-1-carboxamidine.HCl (73 mg, 0.50 mmol) was added to thereaction mixture followed by diisopropylethylamine (87 ml, 0.50 mmol).Dichloromethane was stripped off of the reaction mixture on a rotovapthen rotation continued overnight without vacuum at room temperature.After removing DMF in vacuo the reaction residue was dissolved indichloromethane and applied to a flash silica gel column. A gradient of5 to 20% MeOH/dichloromethane with 0.5 to 2% NH₄OH followed by treatmentwith Dowex OH⁻ (300 mg) in MeOH gave pure (19): 0.11 g, 65%, ³¹P NMR (d,33.83 ppm), mass spec. calcd=676.97, found=677.54.

Example 5 Synthesis of PH 55943

Synthesis of Cholesterol-Lysine-methylphosphonoamidate (PH55943) (20):Referring to FIG. 7, crude (13) was treated with additional DMAP (0.31g, 2.5 mmol) and the reaction mixture cooled to −70° C. while stirringunder positive pressure argon. H-His(Bzl)OCH₃ (0.65 g, 2.5 mmol) inanhydrous dichloromethane (20 ml) was added dropwise via syringe over aperiod of one hour. The reaction mixture was then warmed to roomtemperature and stirred for 18 hours (reaction complete by ³¹P NMR).Direct loading onto flash silica followed by a gradient of 2 to 10%EtOAc/hexanes then 0 to 5% MeOH/dichloromethane gave 0.53 g of (20) (30%over two steps), ³¹P NMR (d, 31.39 ppm), mass spec. calcd=705.96,found-706.47.

Example 6 Synthesis of PH 55945 (21)

Synthesis of Cholesterol-Histidine-methylphosphonamidate (PH55945) (21):Referring to FIG. 8, crude (13) was treated with additional DMAP (0.31g, 2.5 mmol) and the reaction mixture cooled to −70° C. while stirringunder positive pressure argon. 1-(3-aminopropyl)-imidazole (0.30 ml, 2.5mmol) in anhydrous dichloromethane (20 ml) was added dropwise viasyringe over a period of one hour. The reaction mixture was warmed to rtand stirred at rt for an additional 18 hours (reaction complete by ³¹PNMR). Direct loading onto flash silica after saturated bicarb washingfollowed by a gradient of 0 to 10% EtOAc/hexanes then 0 to 10%MeOH/dichloromethane affording 0.77 g of (21): (yield, 54% over twosteps), ³¹P NMR (d, 32.47 ppm), mass spec. calcd=571.82, found=572.33.

Example 7 Synthesis of Vitamin B6 and Beta-Alanine-Based Cationic Lipids

Synthesis of N-CBZ-beta-Alanine N-hydroxysuccinimide ester (22):Referring to FIG. 9, the compound was prepared according to Lewis et al.PNAS 1996, 93, 3176-3181 (incorporated by reference herein). Yield 80%.¹H NMR (DMSO-d₆) d 7.42 (t, 1H, NH), 7.33 (m, 5H, benzyl), 5.009 (s,CH₂), 1.47 (m, 4H), 3.32 (m, 2H, CH₂NH), 2.86 (t, 2H, CH₂CO), 2.79 (s,4H, CH₂CH₂).

Synthesis of N′-palmityl-N′-oleyl-N-CBZ-beta-alanine amide (23):compound 22 (1.0 g, 2.03 mmol) was added to a solution ofN-CBZ-beta-Alanine N-hydroxysuccinimide ester (0.16 g, 0.5 mmol) inCH₂Cl₂ containing Et₃N (0.42 ml, 3 mmol). The reaction mixture wasstirred at room temperature overnight, diluted with dichloromethane andwashed with saturated NaHCO₃ and brine. Organic layer was dried oversodium sulfate and evaporated to dryness. The residue was purified byflash chromatography on silica using mixture of EtOAc-Hexanes (1:3) asan eluent to give 1.32 g of 23 as a yellow oil (yield, 93%). ¹H NMR(CDCl₃) d 7.34 (m, phenyl), 5.62 (t, NH), 5.35 (m, CH═CH), 5.082 (s, 2H,CH₂Ph), 3.49 (m, 2H, CH₂NH), 3.27 (m, 2H), 3.15 (m, 2H), 2.5 (m, 2H,CH₂CO), 2.00 (m, 4H), 1.49 (m, 4H), 1.25 (m, 48H), 0.87 (m, 6H).

Synthesis of N′-palmityl-N′-oleyl-beta-alanine amide (AK 524-68) (POABA)(24): compound 23 (1.2 g, 1.72 mmol) was dissolved in absolute ethanol(10 ml) and 10% Pd—C was added under argon. The flask was then immersedin a 20° C. water bath prior to the addition of 1,4-cyclohexadiene (1.6ml). The reaction mixture was stirred at room temperature for 40 hours,the catalyst was filtered off and the filtrate evaporated to dryness.The residue was purified by flash chromatography on silica eluting withthe linear gradient of MeOH (5% to 10%) in dichloromethane to give 0.68g, of 24 (yield, 70%). ¹H NMR (CDCl₃) d 5.37 (m, 2H, CH═CH), 3.26 (m,4H), 3.16 (m, 2H), 2.75 (t, NH₂), 1.95 (m, 2H), 1.51 (m, 4H), 1.25 (m,48H), 0.87 (m, 6H). FAB-MS: 563.6 [M+H]⁺.

Synthesis of N′-palmityl-N′-oleyl-N-carboxamidine-beta-alanine amide (AK524-73) (GPPOA) (25): The mixture of 24 (60 mg, 0.11 mmol), pyrazolecarboxamidine hydrochloride (16 mg, 0.11 mmol) and diisopropylethylamine(20 mL, 0.12 mmol) in 0.5 mL of THF-MeOH (1:1) was stirred overnight atroom temperature. It was then evaporated to dryness, dissolved indichloromethane and washed with aqueous ammonia. The organic layer wasdried over sodium sulfate to give 25 as an yellowish oil. Yield nearquantitative. ¹H NMR (CDCl₃) d 7.70-7.25 (m, 5H, NH), 5.38 (m, 2H,CH═CH), 3.43 (m, 2H, CH₂NH), 3.23 (m, 2H), 3.17 (m, 2H), 2.54 (m 2H,CH₂CO) 1.96 (m, 4H), 1.49 (m, 4H), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS:605.6 [M+H]⁺.

Synthesis of N(N″-palmityl-N″-oleyl-amidopropyl)pyridoxamine (AK 524-74)(POCAEP) (26): Compound 26 was prepared analogously to compound 27(Yield, 78%). FAB-MS: 714.6 [M+H]⁺.

Example 8 Synthesis of AK524-76 (27)

N-cholesteryl-pyridoxamine (AK524-76) (CCAEP) (27): Referring to FIG.11, The suspension of Pyridoxal hydrochloride (0.1 g, 0.5 mmol) inethanol was brought to pH 7 with 1N NaOH followed by the addition ofaminocholesterol (0.19 g, 0.5 mmol). The pH of the resulting brightyellow solution was adjusted to 8 (1N NaOH) and set aside for 10minutes. Sodium borohydride (20 mg, 0.5 mmol) was then added to thereaction mixture resulting in immediate color disappearance. After 15minutes reaction mixture was acidified (pH 6) with 1N HCl, diluted withdichloromethane and washed with aqueous ammonia and water. The organiclayer was dried over sodium sulfate and evaporated to dryness. Theresidue was purified by flash chromatography on silica using 10% MeOH indichloromethane as an eluent to give 0.25 g (93%) of 27. ¹H NMR (CDCl₃)d 7.78 (s, 1H, H-6 Pyr), 4.58 (s, 2H, CH₂O), 4.06 (AB-quartet 2H, CH₂N),2.4 (s, 3H, 2-CH₃), 2.2-0.24 (m, cholesteryl moiety). FAB-MS: 537.4[M+H]⁺.

Example 9 Synthesis of 2′-Aminouridine-Based Cationic Lipids

2′-Deoxy-2′-(N-Fmoc-beta-alanineamido) uridine (28): Referring to FIG.10, EEDQ (4.2 g, 17 mmol) was added to the solution of 2′-amino-2′-deoxyuridine (4 g, 16.45 mmol) and N-FMOC-beta-alanine (5.1 g, 16.45 mmol) inmethanol and the reaction mixture was boiled for two hours. Subsequentflash chromatography on silica using a linear gradient of methanol (5%to 10%) in dichloromethane afforded 6 g of2′-Deoxy-2′-(N-Fmoc-beta-alanineamido) uridine (77%). ¹H NMR(CDCl₃-DMSO-d6) d 10.398 (s, 1H, N3-H), 6.98-7.63 (m, H6, Fmoc), 6.16(t, 1H, NHFmoc), 5.73 (d, 1H, H1′, J_(1′,2′) 8.4), 5.34 (d, 1H, H5),4.22 (m, 1H, 2′-H) 3.98 (dd 2H, CH₂), 3.88 (m, 1H, 3′-H), 3.77 (br s,1H, 4′-H), 3.43 (m, 2H, 5′-CH₂), 3.1 (m, 2H, CH ₂NHFmoc), 2.07 (t, 2H,CH ₂CO).

Synthesis of 3′,5′-Di-palmitoyl-2′-deoxy-2′-(N-Fmoc-beta-alanineamido)uridine (29): Palmitoyl chloride (1.55 mL, 1.8 mmol) was added to asolution of nucleoside 28 in abs pyridine and the reaction mixture wasstirred overnight at room temperature. The solution was then quenchedwith MeOH, evaporated to dryness, dissolved in dichloromethane andwashed with saturated aq sodium bicarbonate and brine. The organic phasewas dried oversodium sulfate and evaporated to dryness. The residue waspurified by flash chromatography on silica (EtOAc-Hexanes 1:1) affording0.5 g of 29 (yield, 65%).

Synthesis of 3′,5′-Di-palmitoyl-2′-deoxy-2′-(beta-alanineamido) uridine(AK 524-71) (30): To the solution of 29 (0.5 g, 0.49 mmol) indichloromethane (5 mL) was added morpholine (1 mL) and the reactionmixture was stirred at room temperature for 36 hours. Subsequent flashchromatography on silica using linear gradient of methanol (5% to 10%)in dichloromethane afforded 0.22 g of desired product (yield, 56%) of30. FAB-MS: 791.6 [M+H]⁺.

Synthesis of3′,5′-Di-palmitoyl-2′-deoxy-2′-(N-carboxamidine-beta-alanineamido)uridine (AK 524-75) (31): Compound 31 was prepared analogously tocompound 25. Yield 80%. FAB-MS: 833.6 [M+H]⁺.

Example 10 Preparation of Lipid-Based Formulations Including CationicLipids and DOPE

For each cationic lipid, four aqueous suspensions were prepared, threecontaining the fusogenic neutral lipid DOPE (dioleoyl phosphatidylethanolamine), and one containing the cationic lipid only (Table II).For this, the solid cationic lipids were dissolved in chloroform andaliquots transferred to 5 ml glass tubes with Teflon-lined caps. DOPE,also dissolved in chloroform, was then added to individual tubes at 1:1,2:1, or 3:1 molar ratios (ratio of cationic lipid to DOPE). The lipidmix was deposited as a film in the glass tube by evaporating the solventwith a stream of argon. The lipid film was hydrated with water (1 ml permg total lipid) and then resuspended by sonication using a bathsonicator (three or four 15 s treatments with intermittent vortexmixing). The formulations were stored at 4 C until used (usually within8 weeks).

Example 11 Cell Culture and Synthesis of Anionic Polymers

Cellular delivery and efficacy assays were carried out in monolayercultures of cells originating from normal tissues or tumors (Table II,III and V). Cells were maintained in humidified incubators using growthmedium recommended by the supplier. Hammerhead ribozymes weresynthesized and purified using standard protocols (Wincott et al., 1995,23, 2677; Beigelman et al., 1995, J. Biol. Chem. 270, 25702; both areincorporated by reference herein). Nuclease resistant linkages wereincorporated at specific sites of the ribozymes, modifications thatmarkedly increased the serum half-life from a few minutes to severalhours. For cellular delivery studies, fluorophore-tagged 32-merribozymes were prepared by attaching a fluorescein or rhodamine moietyto the loop portion through a aminolinker-modified base (FIG. 13). Anexpression plasmid encoding the humanized Green Fluorescent Protein(plasmid pEGFP-C1) was obtained from Clontech.

Example 12 Cellular Ribozyme Delivery

For the delivery studies, subconfluent cultures of mammalian cells wereseeded in 24-well plates (˜20,000 cells/well) a day prior to theinitiation of assay. In a typical delivery assay, 100 μl of a 1 μMfluorescein or rhodamine-conjugated ribozyme (i.e., 10 X ribozymediluted in water) was placed in a polystyrene tube, and an aliquot ofthe cationic lipid formulation was added at room temperature to allowcomplex formation. The appropriate growth medium added to each tube (0.9ml) and then the contents were mixed and added to each well. Finalconcentration of the ribozyme was 100 nM and the transport vehicleconcentration was varied from 2.5 to 20 μg/ml. After a 3-4 h incubation,the medium was replaced with normal growth medium and the localizationof the cell-associated ribozyme was evaluated by fluorescence microscopyusing a Nikon stage microscope equipped with a 40× objective and a ccdcamera. In some studies, the total cell-associated ribozyme wasquantified by FACS analysis.

Example 13 Cellular Plasmid Delivery

Subconfluent cultures of cells were seeded in 24-well plates (˜10,000cells/well). In typical transfection studies, 100 ng of the plasmid in0.1 ml water was premixed with individual lipids in a polyethylene tubeand incubated at room temperature for ˜10 minutes to allow complexformation. Then 0.9 ml growth medium (serum-free) was added to eachtube, the contents were mixed, and administered to individual wells ofcells for 3-4 h. The medium was then replaced with normal growth mediumand cells were left for ˜24 h. Expression of GFP was monitored byfluorescence microscopy. The transport vehicles that led to GFPexpression in the highest percentage of cells were identified for eachcell line (Table IV).

Example 14 Cytotoxicity Analysis

The toxic effects of the lipid-formulated compositions on cells weredetermined in three ways. First, cellular morphology was evaluated inrelation to normal, untreated cells and significant abnormalities orreduction in cell numbers were noted. Second, for evaluating grosstoxicity, propidium iodide was added to the medium and the cells wereexamined for the presence of pink-red fluorescence in the nucleus,indicating the presence of perforated or damaged membranes. Finally, thelonger term effect of the treatment on cells was quantified using asensitive MTS proliferation assay (Promega).

Example 15 c-myb Proliferation Assay

The protooncogene c-myb is a transcription factor that participates inregulating the proliferation of smooth muscle cells. It has beendemonstrated that cells in which c-myb levels have been reduced byribozyme-Lipofectamine treatment do not proliferate well in response tosubsequent serum stimulation. Two ribozymes directed against c-mybtermed “active” and “inactive” (Jarvis et al., 1995, RNA, 2, 419). Bothribozymes can recognize the mRNA target but only the “active” can cleaveit. The “inactive” ribozyme serves as a negative control. In principle,the active ribozyme can reduce c-myb expression by catalyzing thesequence-specific cleavage of the mRNA, leading to a reduction in cellproliferation. This assay was used to validate the utility of deliveryformulations. 3H-Thymidine Incorporation Assay

In typical cell proliferation, subconfluent cultures of rat aorticsmooth muscle cells (RASMC) were plated in 48-well plates in DMEMsupplemented with amino acids, Hepes, and 10% fetal bovine serum (5000cells/well/0.5 ml medium). Next day, cells were serum-starved, toinhibit proliferation, by replacing the medium with low serum medium(0.5% serum) for ˜2 days. The starved cells were then treated withribozyme-carrier formulations, usually 100 nM ribozyme premixed with2.5-10 μg/ml carrier lipid, in serum-free medium for ˜2 h, followed by“trafficking” in low serum-medium (0.25% serum) for ˜20 h. Triplicateset of wells were exposed to each treatment. The cells were thenstimulated to proliferate for 12 h in medium containing 10% serum. Thiswas followed by another 8 h incubation in medium+10% serum+³H-thymidine(˜1 μCi/ml). The cells were then fixed with ice-cold 10% trichloroaceticacid, washed twice with water, and ³H-thymidine incorporated into newDNA was measured by scintillation counting. The inhibition ofproliferation using different ribozyme formulations is shown in Table V.

Example 16 Cellular Transport of Lipophilic Compounds

Rhodamine-conjugated dioleoyl phosphatidyl ethanolamine (DOPE) was mixedwith various cationic lipids and administered to cells seeded in 24-wellplates. After ˜3 h incubation, the cellular distribution of thefluorescent rhodamine-DOPE was examined by fluorescence microscopy.Every cell contained rhodamine (red fluorescence), indicating that thelipids were delivered efficiently to the cells. Next,fluorescein-conjugated ribozymes were packaged and coadministered tocells using the same vehicles using procedures described earlier. Again,every cell was labeled with rhodamine while a subset containedinternalized ribozymes (green fluorescence). These observationssuggested the lipid transporters can be used to deliver lipophilic aswell as hydrophilic compounds to cells.

Example 17 Delivery of Antisense Molecules

A 21-nucleotide long phosphorothioate oligodeoxynucleotide with anattached fluorescein moiety was synthesized by standard procedures. Theoligonucleotide (100 nM) was formulated with different concentrations(2.5 to 10 μg/ml) of each transport vehicle and administered to cells.Subcellular distribution of the internalized material was evaluated byfluorescence microscopy. The results indicated that optimal transporterconcentrations are different for antisense oligonucleotides compared toribozymes.

Example 18 Synthesis ofN²,N³-di-oleyl-(N,N′-diguanidinoethyl-aminoethane)-2,3-diaminopropionicacid (36)

Referring to FIG. 14, applicant describes a reaction of2,3-diaminopropionic acid 32 with oleoyl chloride in the presence ofdimethylaminopyridine (DMAP) and triethylamine (TEA) can give theperacylated derivative 33, oleyl. Reaction of 33 withtriethylenetetramine (TREN) 34, followed by reaction with1H-Pyrazole-1-carboxamidine hydrochloride 35 can give the title compound36.

Example 19 Preparation of PC:CHOL:DOTAP:DSPE₂₀₀₀ Liposome Formulation

Formation of EPC:CHOL:DOTAP:DSPE-PEG₂₀₀₀: Egg yolk phosphatidylcholine(EPC), cholesterol, and DOTAP were purchased from Avanti Polar Lipids.DSPE-PEG₂₀₀₀(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000) was purchased from Shearwater polymers. Extruder waspurchased from Lipex biomembranes. FPLC was purchased from Pharmacia.Radioactive compounds were purchased from NEN and ether from Sigma.

The following lipids suspended in chloroform were mixed together in a 50ml round bottom flask: phosphatidylcholine (egg yolk) (85.5 mg),cholesterol (21.8 mg), DOTAP (23.7 mg), ceramide-PEG C20 (61.8 mg)resulting in a molar ratio of 50:25:15:10. A tracer of Cholesterylhexadecyl ether (CHE) (26 μCi) was added to monitor lipid concentration.The lipids were dried down by rotary evaporation and then resuspended inether (9 ml). Ribozyme (25 mg) suspended in 1× phosphate buffered saline(3 ml) was added to the ether/lipid mixture and mixed together into anemulsion. The ribozyme was quantitated using a ³²P internally labeledribozyme tracer (160 μCi). Liposome vesicles were formed by removing theether under vacuum. Residual ether was removed by bubbling argon gasthrough the lipid-ribozyme mixture for 10 minutes. Vesicles were thenpassed through polycarbonate filters with 200 nm and 100 nm poresconsecutively 6-10 times using an Extruder (Lipex Biomembranes,Vancouver, B. C.). Liposomes were purified from unencapsulated materialusing an FPLC column packed with DEAE sepharose CL-6B. Ribozyme andlipid concentration was determined by reading an aliquot of purifiedliposomes on a scintillation counter for both tritium and 32P. Thecounts indicated that 5.75 mg of ribozyme was present within liposomevesicles (23% encapsulation).

Example 20 Blood Clearance Study Using the EPC:CHOL:DOTAP:DSPE₂₀₀₀Liposome

Female C57B1/6J weighing 20-25 g were used in this study. with 3 mmol oflipid (36 mg ribozyme) by tail vein injection. The time points observedwere 15 minutes, 1 hour, 4 hour, and 24 hours with 3 mice per group. Theanimals were euthanized by CO₂ asphyxiation. Upon cessation ofbreathing, the chest cavity was opened and blood sampled (200-500 μL)from the heart. Sampled blood was added to a heparinized microfuge tubeand centrifuged for 10 minutes to separate plasma and blood cells.Plasma samples were treated with proteinase K containing buffer (100 mMNaCl, 10 mM tris (pH 8), 25 mM EDTA, 10% SDS). A portion of the samplewas to scintillant and counted. The remaining sample was resolved on apolyacrylamide gel and intact ribozyme bands were quantitated using aphosphorimager (molecular devices). The results are shown in FIG. 15.Formulation of ribozyme with EPC:CHOL:DSPE:PEG C18 greatly enhances thecirculation time of intact ribozyme in plasma. Twenty-four hours afteran intravenous bolus injection of 2 mg/kg ribozyme formulated withEPC:CHOL:DSPE:PEG₂₀₀₀, over 6% of the dose remained in the plasma.Average concentrations dropped from an average of 6631 ng/ml at 15minutes to 2305 ng/ml at 24 hours. Since plasma concentrations wererelatively high 24 hours after an injection, it can be assumed that theelimination half-life is on the order of hours if not days. Incomparison, an intravenous bolus injection of 30 mg/kg is no longerdetectable after approximately 3 hours. The elimination half-life ofunformulated ribozyme is approximately 30 minutes in the mouse.

Example 21 Plasmid DNA Delivery into Cells in Culture

One day prior to transfection, target cells were plated to a finalconfluency of 50 to 60% on a 48 well plate. Cells types tested in serumfree conditions are RT-4 (human bladder carcinoma), EJ (human bladdercarcinoma), PC-3 prostate cancer cell line), and MCF-7 (breast cancercell line). The following cell types were tested in the presence of 10%serum in the media: RT-4, PC-3 and MCF-7 cells. DNA (1 μg of pEGFP-C1C-terminal protein fusion vector (Clontech)) was added to a polystyrenetube followed by the addition of 1 ml of desired media. Followingagitation, the cationic lipid was added to the tube (1.25, 2.5, 5, or 10μg lipid/μg DNA), incubated at room temperature for 15 minutes and thenmixed by vortexing. Media from plated cells was aspirated and thenwashed with either serum free or normal growth media. 200 μL of theDNA/cationic lipid mixture was added to each well of a 48 well plate.The cells were incubated at 37° C. for 3 to 5 hours for serum-freeuptake and 18 to 24 hours for uptake in the presence of serum.Fluorescent cells were then counted using fluorescence microscopy. Thetransfection rate was determined by comparing the number of fluorescencepositive cells to the total number of cells in the microscope field.

Toxicity was determined by adding 5 μL of a 0.5 mg/ml stock solution ofpropidium iodine (PI) (Boehringer Mannheim) prior to examination bymicroscopy. Migration of the red dye into the nucleus of a cellindicated toxicity and loss of cell viability. The results of plasmiddelivery in serum free media are shown in table VI. The results ofplasmid delivery in the presence of serum is shown in table VII.

FACS (fluorescence activated cell sorting) analysis was performed onPC-3 cells using several formulations and the results are shown in tableVIII. Transfection was achieved using the protocol described above withthe cells being incubated with DNA for 4 hours in serum free conditions.Cells were trypsinized off the plate, collected in serum containinggrowth media, and spun down for 5 minutes at 800 RPM. The supernatantwas removed and the cells were brought up in 500 μL of FAC buffer (4%FBS in Hank's balanced salt solution (HBSS)). 10 μL of 0.5 mg/ml of PIwas added prior to FACS sorting. The results indicate that applicant'sformulations improve delivery of macromolecules compared to othercompounds which are commercially available.

Example 22 Preparation of Cationic Lipids Conjugated to PolyethyleneGlycol Via Amide Bond

Cationic lipid (100 mg), methoxypolyoxyethylenecarboxylic acid (725 mg),and 1,3-dicyclohexylcarbodiimide (DCC) (30 mg) were dissolved inchloroform (30 mL) and the solution was allowed to react at 50° C.overnight. The reaction mixture was filtered and hexane was added to thefiltrate for purification by precipitation. The product(N′-palmityl-N′-oleyl-α-amino,γ-PEGamino-glycinamide) wasre-precipitated using the same procedure and then dried in vacuo toobtain a PEG-conjugated lipid. In addition to carboxy-terminated PEG,N-hydroxysuccinimide activated ester of PEG can be utilized in the aboveconjugation procedures.

Conjugation may also be carried out by formation of a carbamate bond.The reaction would be initiated by reacting imidazolylcarbonateactivated hydroxy-terminated methoxypolyethylene glycol with aminogroups of cationic lipids described above. The methods described hereinare not limiting. Those skilled in the art will recognize that PEG canbe readily conjugated to cationic lipids using techniques known in theart and are within the scope of this invention.

Example 23 Transfection of Jurkat Cells with Fluorescein ConjugatedOligonucleotide

The ability of cytofectins of the invention to transfect suspensioncells was tested in Jurkat cells. These cells were grown and maintainedin RPMI-1640 culture medium supplemented with 10% fetal bovine serum andglutamine (Life Technologies). The cells were diluted in culture mediumto 5×10⁵ cells/ml, and 80 μl (40,000 cells) was transferred into eachwell of a 96-well culture dish. A fluorescein-conjugated, nucleaseresistant 23-mer oligonucleotide of randomized sequence was chemicallysynthesized for transfection. Additional oligonucleotides weresynthesized using standard methodologies (Wincott et al., supra).

The fluorescein-conjugated oligonucleotides were premixed withcytofectins (Table IX) at 5× concentrations (20 μl volume) and added towells containing cells in 80 μl medium (total incubation volume=100 μl).In typical experiments, 100 nM oligonucleotide (final concentration) wasmixed with 2.5, 5, or 10 μg/ml of each delivery vehicle. In someexperiments, culture plates were immediately placed in a humidifiedincubator overnight (18-24 h). In other experiments, the platescontaining cells and oligonucleotide-lipid complexes were first spun for40 minutes at room temperature and then incubated overnight. Thefollowing day, the cells were gently spun down and the incubation mediumwas replaced with normal growth medium.

Delivery of oligonucleotides into cells was determined by fluorescencemicroscopy. The cellular nuclei were examined to identify whetherfluorescence was emitted from within the nuclear envelope. Nucleardelivery indicated that the oligonucleotide had permeated across thetopological membrane barrier and entered the cytosol followed bymigration to nucleus. Alternatively, punctate perinuclear pattern offluorescence around the nucleus indicated that the oligonucleotide,internalized by an endocytic mechanism, remained sequestered within theendosomal vesicles. Jurkat cells are small and round and the nucleusoccupies much of the cellular volume. Nuclear as well as perinuclearcytoplasmic labeling was observed in most cells. The efficiency ofdelivery was estimated by calculating the number of cells with greennuclei as a percentage of the total number of cells in the field, asobserved by phase contrast microscopy. The acute toxicity of thetreatments was assessed by adding propidium iodide (1 μg/ml) to eachwell. Damaged cells with compromised membranes internalized propidiumiodide and could be easily identified by red fluorescence in thenucleus.

Three cytofectins (Formulation ID Nos: 345, 323, and 333) were found totransfect the oligonucleotide at a very efficient rate. Depending on theformulation, delivery into the nuclei of jurkat cells ranged from 40 to80% (Table IX).

Example 24 Inhibition of Inosine Monophosphate Dehydrogenase (IMPDH)Using Nucleic Acid Molecules

The cytofectins identified using the delivery screen described inExample 23 were then used in efficacy assays. An antisense moleculedirected against the mRNA for IMPDH, an essential enzyme involved innucleic acid production, was formulated with the delivery vehicle andadministered to Jurkat cells seeded in 96-well plates as describedabove. Assays were done in triplicate wells and cells were incubated for24 h. A random sequence antisense molecule of the same length andcontaining the same type of linkages as the IMPDH binding antisensemolecule was used as control. A quantitative Taqman assay (Perkin Elmer)was developed to measure the changes in IMPDH mRNA levels relative toactin, a constitutively expressed housekeeping gene. RNA from treated oruntreated cells was extracted and then analyzed by Taqman. All three ofthe cytofectins tested were able to deliver sufficient antisensemolecule to inhibit the expression of IMPDH-2 (FIGS. 16-18).

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group. Other embodiments are within the claims thatfollow.

TABLE I Characteristics of naturally occurring ribozymes Group I IntronsSize: ~150 to >1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintainance of the active structure. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies [,¹].Complete kinetic framework established for one ribozyme [²,³,⁴,⁵].Studies of ribozyme folding and substrate docking underway [⁶,⁷,⁸].Chemical modification investigation of important residues wellestablished [⁹,¹⁰]. The small (4-6 nt) binding site may make thisribozyme too non-specific for targeted RNA cleavage, however, theTetrahymena group I intron has been used to repair a “defective”β-galactosidase message by the ligation of new β-galactosidase sequencesonto the defective message [¹¹]. RNAse P RNA (M1 RNA) Size: ~290 to 400nucleotides. RNA portion of a ubiquitous ribonucleoprotein enzyme.Cleaves tRNA precursors to form mature tRNA [¹²]. Reaction mechanism:possible attack by M²⁺-OH to generate cleavage products with 3′- OH and5′-phosphate. RNAse P is found throughout the prokaryotes andeukaryotes. The RNA subunit has been sequenced from bacteria, yeast,rodents, and primates. Recruitment of endogenous RNAse P for therapeuticapplications is possible through hybridization of an External GuideSequence (EGS) to the target RNA [¹³,¹⁴] Important phosphate and 2′ OHcontacts recently identified [¹⁵,¹⁶] Group II Introns Size: >1000nucleotides. Trans cleavage of target RNAs recently demonstrated[¹⁷,¹⁸]. Sequence requirements not fully determined. Reaction mechanism:2′-OH of an internal adenosine generates cleavage products with 3′- OHand a “lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Onlynatural ribozyme with demonstrated participation in DNA cleavage [¹⁹,²⁰]in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [²¹]. Important 2′OH contacts beginning to be identified [²²] Kinetic framework underdevelopment [²³] Neurospora VS RNA Size: ~144 nucleotides. Transcleavage of hairpin target RNAs recently demonstrated [²⁴]. Sequencerequirements not fully determined. Reaction mechanism: attack by 2′-OH5′ to the scissile bond to generate cleavage products with 2′,3′-cyclicphosphate and 5′-OH ends. Binding sites and structural requirements notfully determined. Only 1 known member of this class. Found in NeurosporaVS RNA. Hammerhead Ribozyme (see text for references) Size: ~13 to 40nucleotides. Requires the target sequence UH immediately 5′ of thecleavage site. Binds a variable number nucleotides on both sides of thecleavage site. Reaction mechanism: attack by 2′-OH 5′ to the scissilebond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OHends. 14 known members of this class. Found in a number of plantpathogens (virusoids) that use RNA as the infectious agent. Essentialstructural features largely defined, including 2 crystal structures[²⁵,²⁶] Minimal ligation activity demonstrated (for engineering throughin vitro selection) [²⁷] Complete kinetic framework established for twoor more ribozymes [²⁸]. Chemical modification investigation of importantresidues well established [²⁹]. Hairpin Ribozyme Size: ~50 nucleotides.Requires the target sequence GUC immediately 3′ of the cleavage site.Binds 4-6 nucleotides at the 5′-side of the cleavage site and a variablenumber to the 3′- side of the cleavage site. Reaction mechanism: attackby 2′-OH 5′ to the scissile bond to generate cleavage products with2′,3′-cyclic phosphate and 5′-OH ends. 3 known members of this class.Found in three plant pathogen (satellite RNAs of the tobacco ringspotvirus, arabis mosaic virus and chicory yellow mottle virus) which usesRNA as the infectious agent. Essential structural features largelydefined [³⁰,³¹,³²,³³] Ligation activity (in addition to cleavageactivity) makes ribozyme amenable to engineering through in vitroselection [³⁴] Complete kinetic framework established for one ribozyme[³⁵]. Chemical modification investigation of important residues begun[³⁶,³⁷]. Hepatitis Delta Virus (HDV) Ribozyme Size: ~60 nucleotides.Trans cleavage of target RNAs demonstrated [³⁸]. Binding sites andstructural requirements not fully determined, although no sequences 5′of cleavage site are required. Folded ribozyme contains a pseudoknotstructure [³⁹]. Reaction mechanism: attack by 2′-OH 5′ to the scissilebond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OHends. Only 2 known members of this class. Found in human HDV. Circularform of HDV is active and shows increased nuclease stability [⁴⁰]Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol.(1994), 1(1), 5-7. ¹Lisacek, Frederique; Diaz, Yolande; Michel,Francois. Automatic identification of group I intron cores in genomicDNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17. ²Herschlag,Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymenathermophila ribozyme. 1. Kinetic description of the reaction of an RNAsubstrate complementary to the active site. Biochemistry (1990), 29(44),10159-71. ³Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavageby the Tetrahymena thermophila ribozyme. 2. Kinetic description of thereaction of an RNA substrate that forms a mismatch at the active site.Biochemistry (1990), 29(44), 10172-80. ⁴Knitt, Deborah S.; Herschlag,Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal anUnconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5),1560-70. ⁵Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H.. Amechanistic framework for the second step of splicing catalyzed by theTetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58. ⁶Li, Yi;Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H.. Thermodynamicand activation parameters for binding of a pyrene-labeled substrate bythe Tetrahymena ribozyme: docking is not diffusion-controlled and isdriven by a favorable entropy change. Biochemistry (1995), 34(44),14394-9. ⁷Banerjee, Aloke Raj; Turner, Douglas H.. The time dependenceof chemical modification reveals slow steps in the folding of a group Iribozyme. 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Conversion of a Group II Intron into a NewMultiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides:Elucidation of Reaction Mechanism and Structure/Function Relationships.Biochemistry (1995), 34(9), 2965-77. ¹⁹Zimmerly, Steven; Guo, Huatao;Eskes, Robert; Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M.. Agroup II intron RNA is a catalytic component of a DNA endonucleaseinvolved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4),529-38. ²⁰Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J.,Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNAlinkages with similar efficiency, and lack contacts with substrate2′-hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70. ²¹Michel,Francois; Ferat, Jean Luc. Structure and activities of group II introns.Annu. Rev. Biochem. (1995), 64, 435-61. ²²Abramovitz, Dana L.; Friedman,Richard A.; Pyle, Anna Marie. Catalytic role of 2′-hydroxyl groupswithin a group II intron active site. Science (Washington, D. 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Biochemistry, (1994) 33, 3374-3385.Beigelman, L., et al., Chemical modifications of hammerhead ribozymes.J. Biol. Chem., (1995) 270, 25702-25708. ²⁹Beigelman, L., et al.,Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995)270, 25702-25708. ³⁰Hampel, Arnold; Tritz, Richard; Hicks, Margaret;Cruz, Phillip. ‘Hairpin’ catalytic RNA model: evidence for helixes andsequence requirement for substrate RNA. Nucleic Acids Res. (1990),18(2), 299-304. ³¹Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke,John M.. Novel guanosine requirement for catalysis by the hairpinribozyme. Nature (London) (1991), 354(6351), 320-2. ³²Berzal-Herranz,Alfredo; Joseph, Simpson; Chowrira, Bharat M.; Butcher, Samuel E.;Burke, John M.. Essential nucleotide sequences and secondary structureelements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73.³³Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.;Butcher, Samuel E.. Substrate selection rules for the hairpin ribozymedetermined by in vitro selection, mutation, and analysis of mismatchedsubstrates. Genes Dev. (1993), 7(1), 130-8. ³⁴Berzal-Herranz, Alfredo;Joseph, Simpson; Burke, John M.. In vitro selection of active hairpinribozymes by sequential RNA-catalyzed cleavage and ligation reactions.Genes Dev. (1992), 6(1), 129-34. ³⁵Hegg, Lisa A.; Fedor, Martha J..Kinetics and Thermodynamics of Intermolecular Catalysis by HairpinRibozymes. Biochemistry (1995), 34(48), 15813-28. ³⁶Grasby, Jane A.;Mersmann, Karin; Singh, Mohinder; Gait, Michael J.. Purine FunctionalGroups in Essential Residues of the Hairpin Ribozyme Required forCatalytic Cleavage of RNA. Biochemistry (1995), 34(12), 4068-76.³⁷Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman,Nassim; Sorensen, Ulrik S.; Gait, Michael J.. Base and sugarrequirements for RNA cleavage of essential nucleoside residues ininternal loop B of the hairpin ribozyme: implications for secondarystructure. 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TABLE II Cationic Lipid Formulations and Cellular Uptake CationicNuclear Compound Cationic Lipid lipid:DOPE Formulation Dose LocalizationCytoplasmic Name Name (molar ratio) Name (μg/ml) 0 = none; 5 = allLocalization DS46596a Arg-chol-2 1:0 nc99 5 1 punct. BOCs 10 1 punct.1:1 nc25 5 2-3 punct. 10 3-4 punct. 2:1 nc26 5 2-3 punct. 10 3 punct.3:1 nc27 5 2-4 punct. 10 2-4 punct. DS46596b Arg-chol-3 1:0 nc100 5 1some clumps BOCs 10 1 some clumps 1:1 nc4 5 4 punct. 10 3 punct. 2:1 nc55 1 punct. 10 1 punct. 3:1 nc6 5 1 punct. 10 1 punct. PHF55933Chol-methyl- 1:1 nc13 5 0 bright punct. 10 0 very bright 2:1 nc14 5 0-1bright punct. 10 0 bright punct. 3:1 nc15 5 0 bright punct. 10 0 verybright AK52450 Dimyristoyl- 1:0 nc108 5 0 few clumps pyridoxal 10 0 1:1nc16 5 0 10 0 2:1 nc17 5 0 10 0 3:1 nc18 5 0 10 0 JA59311 Palmitoyl 1:0nc101 5 0-1 some punct. oleoyl 10 3 punct. glycyldiamino 1:1 nc19 5 2some punct. butyric acid 10 1 bright punct 2:1 nc20 5 1, faint punct. 103, var. punct. bright. 3:1 nc21 5 3 bright punct. 10 3 bright punct.PH55938 Lys-chol. 1:0 nc104 5 0-1, few brt. no punct. 10 0-1 clumps. 1:1nc22 5 0-1 bright punct 10 0-1 bright punct 2:1 nc23 5 0 bright punct 100 bright punct 3:1 nc24 5 0 punctate 10 0 bright punct. AK52465 N- 1:0nc109 5 0-1, very cholesteryl- faint pyridoxamine 10 1, very some clumpsfaint 1:1 nc28 5 0 10 0-1 2:1 nc29 5 0 10 0 3:1 nc30 5 0 10 0 AK52468Beta-alanine 1:0 nc110 5 3, bright punct. palm.oleoyl- 10 3-4, brightpunct. amide 1:1 nc31 5 0 punct. 10 0 punct. 2:1 nc32 5 0-1 punct. 102-3 punct. 3:1 nc33 5 0-1 punct. 10 3-4 punct. AK52471 dipalmitoyl- 1:0nc111 5 0 punct. deoxy- 10 0-1 punct./clumps aminoethyl- 1:1 nc34 5 0clumps carboxamido- 10 0 clumps Uridine 2:1 nc35 5 0 clumps 10 0 clumps3:1 nc36 5 0 clumps 10 0 clumps AK52474 palmitoyl- 1:0 nc112 5 0 oleoyl-10 0 carboxamido- 1:1 nc37 5 0 faint punct. ethyl- 10 0-1 faint punct.pyridoxamine 2:1 nc38 5 0 faint 10 0 faint punct 3:1 nc39 5 0 10 0 faintpunct. PH55939 1:0 nc105 5 0-1 punct. 10 0-1 punct. 1:1 nc40 5 0 punct.10 0 punct. 2:1 nc41 5 0-1 punct/vac. 10 2-3 punct 3:1 nc42 5 0-1 punct.10 0-1 bright punct. PH55941 1:0 nc106 5 0-1 punct., clumps 10 0-1 brt.pnc., clumps 1:1 nc43 5 0-1 punct. 10 2-3 heavy punct. 2:1 nc44 5 0-1heavy punct. 10 0-1 heavy punct. 3:1 nc45 5 0-1 punct., clumps 10 0-1punct., clumps PH55942 1:0 nc107 5 0-1 bright punct. 10 0-1 brightpunct. 1:1 nc46 5 0-1 punct. 10 3 punct. 2:1 nc47 5 0-1 punct. 10 0-1bright punct. 3:1 nc48 5 0-1 punct. 10 0-1 bright punct. JA59312plamitoyl 1:0 nc102 5 1 bright punct. oleoyl 10 4 bright punct.glycylamino- 1:1 nc49 5 3 punct. guanyl 10 4 bright punct. diamino- 2:1nc50 5 3-4 bright punct. butyric acid 10 4 bright punct. 3:1 nc51 5 4-5bright punct. 10 5 bright punct. JA59314 1:0 nc103 5 0-1 punct. 10 0-1heavy punct. 1:1 nc52 5 0 punct. 10 0 punct. 2:1 nc53 5 0 punct. 10 0punct. 3:1 nc54 5 0 faint punct. 10 0 faint punct. AK52475 dipalmitoyl-1:0 nc96 5 0-1 some clumps deoxy- 10 0-1 clumps guanidino- 1:1 nc84 5 0clumps/st. to ethyl- gl. carboxamido- 10 0 clumps/st. to uridine gl. 2:1nc85 5 0 clumps/st. to gl. 10 0 clumps/st. to gl. 3:1 nc86 5 0clumps/st. to gl. 10 0 clumps/st. to gl. JA59316 palmityl- 1:0 nc97 5 0heavy punct. oleoyl- 10 0 heavy punct. diamino- 1:1 nc87 5 0 lt. butyricacid punct./sticky 10 0 lt. punct./sticky 2:1 nc88 5 0 lt. punct./sticky10 0 lt. punct./sticky 3:1 nc89 5 0 sticky/out. 10 0 sticky/out JA59317palmity- 1:0 nc98 5 0-1 faint punct. oleoyl- 10 0-1 punct. guanidine 1:1nc90 5 1-2 punct. 10 2-3 punct. 2:1 nc91 5 1-2 punct. 10 1-2 punct. 3:1nc92 5 1 punct. 10 1 punct.

TABLE III Optimal cationic lipids Cells Description (>50% nuclearuptake) HS27 human foreskin fibroblasts nc49, nc50 HUVEC human umbilicalvein nc51, nc26 endothelial RAOSMC rat aortic smooth muscle nc102, nc49SK-N-SH human neuroblastoma nc21, nc25 EJ human bladder carcinoma nc21,nc49 RT-4 human bladder carcinoma nc49, nc21 MCF-7 human breastcarcinoma nc21 FEM human melanoma nc21 1205 human melanoma nc49 PC-3human prostate carcinoma nc49, nc21 LN-CAP human prostate carcinomanc110, nc21Table III: Lipid-mediated delivery of ribozymes to various cell lines.Cells were treated with 100 nM fluorescein-conjugated ribozymesformulated with a panel of cationic lipids (selected from nc21, nc25,nc26, nc49, nc51, nc102, nc110; see Methods). Subcellular distributionof the ribozyme was determined by fluorescence microscopy. Presence offluorescence in the nucleus indicated that the ribozyme had beentransported across the cell membrane (unconjugated fluorescein does notremain in the nucleus). Lipid formulations that led to high nucleardelivery with no significant toxicity are shown.

TABLE IV Optimal cationic lipids Cells Description (% of cells with GFP)SK-N-SH human neuroblastoma nc49 (40%) nc101 (25%) nc110 (20%) nc32(15%) RASMC rat aortic smooth muscle nc110 (~5%) RT-4 human bladdercarcinoma nc101 (60%) nc19 (20%) nc110 (20%) HS27 human foreskinfibroblasts nc101 (~5%)Table IV: Lipid-mediated delivery of plasmids to various cell lines.Cells were treated with 0.1-1 μg/ml of a green fluorescent protein (GFP)expression plasmid formulated with 2.5-15 μg/ml of selected lipidformulations (Table II), as described (Methods). The expression of GFPwas monitored by fluorescence microscopy ˜20 hours after transfection.Formulations that resulted in GFP expression by ˜5% or more of the cellsare indicated.

TABLE V Lipid (formulation) Nuclear Uptake? % Inhibition JA59312 (nc102)Y > 50% 40% JA59312 (nc49) Y > 40% 38% JA59317 (nc98) Y > 10% 23%JA59311 (nc101) Y > 5% 21% JA59311 (nc20) Y > 10% 0% PH55942 (nc48) N(punct.) 18% AK52468 (nc33) N (punct.) 16% JA59316 (nc97) N (punct.) 0%PHF55933 (nc13) N 0% PZH55938 (nc22) N 0%Table V. Inhibition of cell proliferation by different ribozymeformulations and correlation with cellular and nuclear delivery. Ananti-myb ribozyme and its inactive version (control) were formulatedwith various lipids (Table II) and administered to rat smooth musclecells as described (Methods). The relative activity of the active vs.inactive ribozyme is shown (% inhibition of proliferation). In parallelexperiments, cells were treated with identical formulations of afluorescein-conjugated ribozyme and its subcellular localization wasobserved by fluorescence microscopy (Y, nuclear delivery (% of positivecells); N, no visible nuclear delivery; punct., punctate cytoplasmicfluorescence). In general, formulations that led to improved delivery ofthe ribozyme to the cell and the nucleus also led to increased efficacy.

TABLE VI Delivery of Green Fluorescent Protein Containing Plasmid intoCells in Culture Transfection Cell type Formulation name Compounds rateOptimal lipid dose Toxicity PC-3 nc 19 JA 59311:DOPE at 1:1 40-64% 2.5μg/ml <10% PC-3 nc 20 JA 59311:DOPE at 2:1 40-50% 2.5 μg/ml  <5% PC-3 nc101B JA 59311 70-75%   5 μg/ml <10% PC-3 nc 102 JA 59312 30-45% 2.5μg/ml  10% PC-3 nc 110D AK 52468 60% 2.5 μg/ml  5% PC-3 nc 122 JA 59317& JA 59311-2:1 55-65% 2.5 μg/ml  5-13% PC-3 nc 123 JA 59317 & JA59311-3:1 30-50% 2.5 μg/ml  5% PC-3 nc 128 JA 59317 & PH 55942-2:135-70% 2.5 μg/ml <3-15% PC-3 nc 144 JA 59311 & JA 59312-2:1 40-60% 2.5μg/ml <3-10% PC-3 nc 145 JA 59311 & JA 59312-3:1 50-70% 2.5 μg/ml <3-20%PC-3 nc 146B JA 59311 & AK 52468-1:1 40-69% 2.5 μg/ml <3-8%  PC-3 nc148B JA 59311 & AK 52648-3:1 25-70% 2.5 μg/ml  3-20% PC-3 nc 156B JA59312 & JA 59311 at 3:1 40-65% 1.5 μg/ml  <5% PC-3 nc 168 AK 52468 & JA59312 at 2:1 33-75% 2.5 μg/ml  5-10% PC-3 nc 169 AK 52468 & JA 59312 at3:1 50-79% 2.5 μg/ml  5-10% RT-4 nc 101B JA 59311 30 to 68%   5 μg/ml<10% RT-4 nc 121 JA 59311 & PH 55942-3:1  8-30%   5 μg/ml  <5% RT-4 nc122 JA 59317 & JA 59311-2:1 20% 2.5 μg/ml  <6% RT-4 nc 144 JA 59311 & JA59312-2:1 50%  10 μg/ml <10% RT-4 nc 145 JA 59311 & JA 59312-3:1 50%  10μg/ml <10% EJ cells nc 19 JA 59311:DOPE at 1:1 40%   5 μg/ml 10% EJcells nc 20 JA 59311:DOPE at 2:1 20%   5 μg/ml  5-10% EJ cells nc 101BJA 59311 25-35%  10 μg/ml  5% EJ cells nc 110D AK 52468 30%   5 μg/ml <5% EJ cells nc 122 JA 59317 & JA 59311-2:1 35%   5 μg/ml  10% EJ cellsnc 144 JA 59311 & JA 59312-2:1 40% 2.5 μg/ml  <3% EJ cells nc 145 JA59311 & JA 59312-3:1 35%   5 μg/ml  5% MCF-7 nc 110D AK 52468 20-30% 2.5μg/ml  4-20% MCF-7 nc 121 JA 59311 & PH 55942-3:1 10-40%   5 μg/ml 8-10% MCF-7 nc 146B JA 59311 & AK 52468 at 1:1 50%   5 μg/ml  15% COS-7nc21 JA59311:DOPE (3:1) 40% 2.5 μg/ml  <5% COS-7 nc 101 JA 59311 50%   5μg/ml  <5% COS-7 nc 110 AK 52468 40%   5 μg/ml <5% HeLa nc 145 JA 59311& JA 59312 (3:1) 40-50% 2.5 μg/ml  <5% RT-4 nc 193 JA 59311-1:Tween80 at1:1  5-40%  10 μg/ml <1-40% RT-4 nc 194 JA 59311-1:Tween 80 at 2:110-20%  10 μg/ml <1-10% RT-4 nc 195 JA 59311-1:Tween80 at 3:1 10-40%  10μg/ml  5-10% RT-4 nc 196 JA 59311-1:Tween80 at 4:1 20%   5 μg/ml  <3%RT-4 nc 220 JA 59349:Tween80 at 6:1 15-40%  10 μg/ml  5-10% PC-3 nc 110DAK 52468 40%   5 μg/ml  8% PC-3 nc 194 JA 59311-1:Tween 80 at 2:1 20-45% 10 μg/ml <1-10% PC-3 nc 195 JA 59311-1:Tween80 at 3:1 60%  10 μg/ml 10% PC-3 nc 218 JA 59311-1:Tween80 at 3:1 40-70%  10 μg/ml  5-40% PC-3nc 219 JA 59349:Tween80 at 4:1 60-75%  10 μg/ml 10-20% MCF-7 nc 110D AK52468 50% 2.5 μg/ml  20%

TABLE VII Delivery of Green Fluorescent Protein Containing Plasmid intoCells in Culture in the Presence of 10% serum Formulation Lipid DosePlasmid DNA % GFP positive Lipofectamine* 1.25 μg/ml 1 μg/ml 3  2.5μg/ml 1 μg/ml 24 PFX-6* 1.25 μg/ml 1 μg/ml 4  2.5 μg/ml 1 μg/ml 15 nc146 1.25 μg/ml 1 μg/ml 40  2.5 μg/ml 1 μg/ml 65 nc 148 1.25 μg/ml 1μg/ml 41  2.5 μg/ml 1 μg/ml 56 nc 156 1.25 μg/ml 1 μg/ml 45  2.5 μg/ml 1μg/ml 71 nc 169 1.25 μg/ml 1 μg/ml 53  2.5 μg/ml 1 μg/ml 72*commercially available cationic lipidsTable VIII. FACS Analysis of Plasmid Delivery into PC-3 Cells of SeveralCationic Lipids after 4 Hours in Serum Free Media.

-   GFP=Green Fluorescent Protein.

TABLE IX Lipid Formulations Formulation Lipid ID No. ID No. ratios Lipidname ratios mass ratios 282 700/747 JA59311/chol-linoleate 3/1 283700/747 JA59311/chol-linoleate 1/1 284 726/743 DPPE/JA73852 1/2 285709/727 DS46596a/DEPE 3/1 286 719/726 AK52468/DPPE 1/1 287 727/749DEPE/EP-G-DABA mix 1/1 288 743/747 JA73852/chol-linoleate 1/1 289701/745 JA59312/cholesterol 2/1 290 700/746 JA59311/chol-linolelaidate2/1 291 705/722 JA59396/DOPE 2.98/1   292 732/747 PH55942/chol-linoleate1/1 293 722/742 DOPE/JA73851 1/3 294 700/727 JA59311/DEPE 2/1 295700/722/745 JA59311/DOPE/cholesterol 1/1/1 296 700/705/722JA59311/JA59396/DOPE 9.01/1/3.36 297 701/746 JA59312/chol-linolelaidate2/1 298 700/727 JA59311/DEPE 1/1 299 742/745 JA73851/cholesterol 1/1 300705/726 JA59396/DPPE 1/1 301 709/725 DS46596a/DLPE 3/1 302 726/732DPPE/PH55942 1/2 303 723/736 Tween 80/JA59350 1/1 304 723/736 Tween80/JA59350 1/6 305 701/727 JA59312/DEPE 3.08/1   306 727/742DEPE/JA73851 1/2 307 743/746 JA73852/chol-linolelaidate 3/1 308 701/727JA59312/DEPE 2.06/1   309 726/744 DPPE/JA73853 1/1 310 725/750DLPE/EP-G-AGBA mix   1/2.05 311 722/743 DOPE/JA73852 1/3 312 700/726JA59311/DPPE 2.99/1   313 727/749 DEPE/EP-G-DABA mix 1/2 314 701/745JA59312/cholesterol 3/1 315 705/725 JA59396/DLPE 1/1 316 725/732DLPE/PH55942 1/1 317 727/744 DEPE/JA73853 1/1 318 723/736 Tween80/JA59350 1/2 319 744/746 JA73853/chol-linolelaidate 3/1 320 723/737Tween 80/JA59351 1/1 321 727/743 DEPE/JA73852 1/2 322 701/746JA59312/chol-linolelaidate 1/1 323 726/749 DPPE/EP-G-DABA mix   1/2.99324 709/747 DS46596a/chol-linoleate 1/1 325 723/737 Tween 80/JA59351 1/2326 701/746 JA59312/chol-linolelaidate 3/1 327 705/725 JA59396/DLPE 2/1328 732/747 PH55942/chol-linoleate 2/1 329 701/747JA59312/chol-linoleate 2/1 330 700/726/747 JA59311/DPPE/chol-linoleate3/1/1 331 743/747 JA73852/chol-linoleate 3/1 332 705/722 JA59396/DOPE1/1 333 701/726 JA59312/DPPE 2.06/1   334 705 JA59396 1 335 727/732DEPE/PH55942 1/3 336 722/744 DOPE/JA73853 1/1 337 709/747DS46596a/chol-linoleate 2/1 338 700/726 JA59311/DPPE 2/1 339 723/737Tween 80/JA59351 1/6 340 742/746 JA73851/chol-linolelaidate 3/1 341727/750 DEPE/EP-G-AGBA mix   1/2.06 342 700/705 JA59311/JA59396 1/9 343700/705 JA59311/JA59396 8/2 344 701/725 JA59312/DLPE 2.05/1   345726/749 DPPE/EP-G-DABA mix 1/2 346 725/750 DLPE/EP-G-AGBA mix   1/1.03347 743/747 JA73852/chol-linoleate 2/1 348 742/747JA73851/chol-linoleate 2/1 349 709/727 DS46596a/DEPE 1/1 350 700/722/745JA59311/DOPE/cholesterol 3/1/1 351 744/747 JA73853/chol-linoleate 2/1352 700/745 JA59311/cholesterol 2/1 353 722/743 DOPE/JA73852 1/1 354719/727 AK52468/DEPE 1/1 355 700/705 JA59311/JA59396 2/8 356 727/750DEPE/EP-G-AGBA mix   1/1.03 357 726/749 DPPE/EP-G-DABA mix 1/1 358742/745 JA73851/cholesterol 3/1 359 723/738 Tween 80/JA59352 1/1 360705/726 JA59396/DPPE 2.98/1   361 701/726 JA59312/DPPE 3.09/1   362709/727 DS46596a/DEPE 2/1 363 701/726 JA59312/DPPE 1.03/1   364 701/747JA59312/chol-linoleate 1/1 365 705/722 JA59396/DOPE 3/1 366 723/739Tween 80/JA59353 1/1 367 723/739 Tween 80/JA59353 1/6 368 744/747JA73853/chol-linoleate 1/1 369 709/747 DS46596a/chol-linoleate 3/1 370700/726/745 JA59311/DPPE/cholesterol 1/1/1 371 732/745PH55942/cholesterol 2/1 372 705/726 JA59396/DPPE 2/1 373 705/722JA59396/DOPE 2/1 374 742/747 JA73851/chol-linoleate 3/1 375 701/745JA59312/cholesterol 1/1 376 726/743 DPPE/JA73852 1/1 377 709/726DS46596a/DPPE 3/1 378 723/736 Tween 80/JA59350 1/4 379 719/725AK52468/DLPE 2/1 380 709/726 DS46596a/DPPE 1/1 381 700/727 JA59311/DEPE2.99/1   382 700/705 JA59311/JA59396 6/4 383 700/705/722JA59311/JA59396/DOPE 8.01/2/3.36 384 744/747 JA73853/chol-linoleate 3/1385 742/747 JA73851/chol-linoleate 1/1 386 705/727 JA59396/DEPE 3/1 387700/747 JA59311/chol-linoleate 2/1 388 701/747 JA59312/chol-linoleate3/1 389 726/743 DPPE/JA73852 1/3 390 732/747 PH55942/chol-linoleate 3/1391 700/705/722 JA59311/JA59396/DOPE 5.01/5/3.36 392 719/726AK52468/DPPE 3/1 393 719/726 AK52468/DPPE 2/1 394 705/727 JA59396/DEPE1/1 395 700/726/745 JA59311/DPPE/cholesterol 3/1/1 396 700/725JA59311/DLPE 2.01/1   397 700/705 JA59311/JA59396 1/1 398 700/705/722JA59311/JA59396/DOPE 6.01/4/3.36 399 742/746 JA73851/chol-linolelaidate1/1 400 700/705/722 JA59311/JA59396/DOPE 7.01/3/3.36 401 709/726DS46596a/DPPE 2/1 402 744/745 JA73853/cholesterol 1/1 403 722/742DOPE/JA73851 1/2 404 744/745 JA73853/cholesterol 2/1 405 722/744DOPE/JA73853 1/2 406 723/738 Tween 80/JA59352 1/6 407 700/725JA59311/DLPE 1/1 408 725/750 DLPE/EP-G-AGBA mix   1/3.09 409 709/725DS46596a/DLPE 2/1 410 725/732 DLPE/PH55942 1/2 411 700/705JA59311/JA59396 4/6 412 700/705/722 JA59311/JA59396/DOPE 3/7/3.35 413700/705/722 JA59311/JA59396/DOPE 2/8/3.36 414 700/705/722JA59311/JA59396/DOPE 1.31/9/3.36 415 723/738 Tween 80/JA59352 1/4 416727/744 DEPE/JA73853 1/2 417 719/725 AK52468/DLPE 1/1 418 700/745JA59311/cholesterol 3/1 419 726/732 DPPE/PH55942 1/1 420 727/732DEPE/PH55942 1/2 421 744/745 JA73853/cholesterol 3/1 422 709/745DS46596a/cholesterol 3/1 423 705/727 JA59396/DEPE 2/1 424 727/743DEPE/JA73852 1/3 425 701/725 JA59312/DLPE 3.09/1   426 727/732DEPE/PH55942 1/1 427 723/738 Tween 80/JA59352 1/2 428 743/745JA73852/cholesterol 2/1 429 743/746 JA73852/chol-linolelaidate 2/1 430700/705 JA59311/JA59396 3/7 431 700/746 JA59311/chol-linolelaidate 3/1432 722/743 DOPE/JA73852 1/2 433 727/750 DEPE/EP-G-AGBA mix   1/3.08 434700/725 JA59311/DLPE 3/1 435 732/745 PH55942/cholesterol 3/1 436 727/743DEPE/JA73852 1/1 437 743/745 JA73852/cholesterol 3/1 438 719/727AK52468/DEPE 2.99/1   439 725/732 DLPE/PH55942 1/3 440 725/749DLPE/EP-G-DABA mix 1/3 441 743/746 JA73852/chol-linolelaidate 1/1 442743/745 JA73852/cholesterol 1/1 443 726/744 DPPE/JA73853 1/3 444 727/742DEPE/JA73851 1/3 445 744/746 JA73853/chol-linolelaidate 2/1 446 701/725JA59312/DLPE 1.03/1   447 705/725 JA59396/DLPE 2.99/1   448 709/745DS46596a/cholesterol 1/1 449 700/705 JA59311/JA59396 9/1 450 709/725DS46596a/DLPE 1/1 451 742/745 JA73851/cholesterol 2/1 452 725/749DLPE/EP-G-DABA mix   1/2.01 453 726/742 DPPE/JA73851 1/3 454 726/744DPPE/JA73853 1/2 455 722/742 DOPE/JA73851 1/1 456 732/745PH55942/cholesterol 1/1 457 726/742 DPPE/JA73851 1/1 458 700/746JA59311/chol-linolelaidate 1/1 459 700/705 JA59311/JA59396 7/3 460 700JA59311 1 461 725/749 DLPE/EP-G-DABA mix 1/1 462 727/749 DEPE/EP-G-DABAmix   1/2.99 463 719/725 AK52468/DLPE 3/1 464 727/744 DEPE/JA73853 1/3465 700/705/722 JA59311/JA59396/DOPE 4.06/6/3.4 466 700/722 JA59311/DOPE2/1 467 744/746 JA73853/chol-linolelaidate 1/1 468 723/739 Tween80/JA59353 1/2 469 727/742 DEPE/JA73851 1/1 470 723/739 Tween 80/JA593531/4 471 723/737 Tween 80/JA59351 1/4 472 700/722/745JA59311/DOPE/cholesterol 2/1/1 473 700/726/747JA59311/DPPE/chol-linoleate 2/1/1 474 726/732 DPPE/PH55942 1/3 475726/742 DPPE/JA73851 1/2 476 700/745 JA59311/cholesterol 1/1 477 719/727AK52468/DEPE 2/1 478 700/726/745 JA59311/DPPE/cholesterol 2/1/1 479700/726 JA59311/DPPE 1/1 480 700/722 JA59311/DOPE 2.99/1   481 722/744DOPE/JA73853 1/3 482 701/727 JA59312/DEPE 1.03/1   483 700/722JA59311/DOPE 1/1 484 709/745 DS46596a/cholesterol 2/1 485 700/726/747JA59311/DPPE/chol-linoleate 1/1/1 486 719/752 AK52468/JA94882 1/2 487700/722 JA59311/DOPE 2/1 488 751/752 JA94881/JA94882 1/1 489 701/726JA59312/DPPE 1.5/1   490 751/752 JA94881/JA94882 0.2/1   491 752/753JA94882/JA10334 0.5/1   492 751/753 JA94881/JA10334 1/1 493 726/751DPPE/JA94881 1/2 494 701/726 JA59312/DPPE 1.8/1   495 751/753JA94881/JA10334 0.5/1   496 719/752 AK52468/JA94882 1/3 497 751/753JA94881/JA10334 2/1 498 700/722 JA59311/DOPE 1/1 499 700/722JA59311/DOPE 3/1 500 753 JA10334 1 501 726/752 DPPE/JA94882 1/2 502719/751 AK52468/JA94881 1/2 503 751/753 JA94881/JA10334 0.2/1   504700/726 JA59311/DPPE 2.4/1   505 700/726 JA59311/DPPE 1.5/1   506719/752 AK52468/JA94882 2/1 507 752/753 JA94882/JA10334 1/1 508 701/726JA59312/DPPE 2.3/1   509 719/752 AK52468/JA94882 3/1 510 751 JA94881 1511 719/751 AK52468/JA94881 1/3 512 719/751 AK52468/JA94881 1/1 513752/753 JA94882/JA10334 0.2/1   514 752/753 JA94882/JA10334 2/1 515751/753 JA94881/JA10334 5/1 516 726/751 DPPE/JA94881 1/3 517 751/752JA94881/JA94882 0.5/1   518 726/751 DPPE/JA94881 1/1 519 719/752AK52468/JA94882 1/1 520 751/752 JA94881/JA94882 5/1 521 722/751DOPE/JA94881 1/1 522 726/752 DPPE/JA94882 1/3 523 726/752 DPPE/JA948821/1 524 700/726 JA59311/DPPE 1.9/1   525 700/726 JA59311/DPPE 3/1 526701/726 JA59312/DPPE 1.2/1   527 752 JA94882 1 528 719/751AK52468/JA94881 2/1 529 722/751 DOPE/JA94881 1/3 530 751/752JA94881/JA94882 2/1 531 719/751 AK52468/JA94881 3/1 532 722/751DOPE/JA94881 1/2 533 752/753 JA94882/JA10334 5/1

TABLE X Lipid Components Names and Structures Lipid_ID Name AlternateNames 700 JA59311 Palmityloleyl glycyl 2,4-diaminobutyric acid; POGDABA;C₁₆C₁₈GlyDABA²⁺; JA59311

701 JA59312 JA59312; Palmityloleyl glycyl 2-amino-4-guanylbutyric acid;POGAGuaBA; C₁₆C₁₈GlyA(Gua)BA²⁺

705 JA59396 N′-Elaidyl-N′-palmityl-a,g-diaminobutyryl-glycinamide;C₁₆C_(18:1)GlyDABA⁺²; JA59396

709 DS46596a BocArgChol; DS46596a

719 AK52468 b-Alanine Palmitoyl, Oleoyl-Amide; POABA; AK52468

722 DOPE DOPE; dioleoyl phosphatidyl ethanolamine;1,2-Dioloeyl-sn-Glycero-3-Phosphatidylethanolamine

723 Tween 80 Tween 80; polyoxyethylene sorbitanmonooleate

725 DLPE DLPE; 1,2-Dilinoeyl-sn-Glycero-3-Phosphatidylethanolamine;Dilinoeyl Phosphatidylethanolamine

726 DPPE DPPE; 1,2-Diphytanoyl-sn-Glycero-3-Phosphatidylethanolamine;Diphytanoyl Phosphatidylethanolamine

727 DEPE DEPE; 1,2-Dielaidoyl-sn-Glycero-3-Phosphatidylethanolamine;Dielaidoyl Phosphatidylethanolamine

732 PH55942 PZH559-42; Cholesterol-TREN-bis-guanidiniummethylphosponamidate

736 JA59350 JA59350;N′-palmityl-N′-oleyl-N-α-Boc-N-γ-carboxamidine-α,γ-diaminobutyryl-glycinamide

737 JA59351 JA59351;N′-palmityl-N′-oleyl-γ-Boc-α,γ-diaminobutyryl-glycinamide

738 JA59352 JA59352;N′-palmityl-N′-oleyl-N-γ-Boc-N-α-carboxamidine-α,γ-diaminobutyryl-glycinamide

739 JA59353 JA59353;N′-palmityl-N′-oleyl-N-γ-carboxamidine-α,γ-diaminobutyryl-glycinamide

742 JA73851 N-(L-Histidyl)-L-a-Dioleyl Phosphatidylethanolamine;His-DOPE

743 JA73852 N′-Palmityl-N′-Oleyl-4-Imidazoleacetyl Glycinamide

744 JA73853 N′-Palmityl-N′-Oleyl-4-(dimethylamino)butyrylamide

745 cholesterol cholesterol;5-Cholesten-3-beta-ol-3-beta-hydroxy-5-cholestene

746 chol-linolelaidate chol-linolelaidate; cholesterol-linolelaidate

747 chol-linoleate chol-linoleate; cholesterol-linoleate; cholesteryl9,12-octadecadienoate; 5-cholesten-3-beta-ol-3-linoleate

749 EP-G-DABA mix N′-Elaidyl-N′-palmityl-α,γ-diaminobutyryl-glycinamide;C₁₆C_(18:1)GlyDABA⁺²; EP-G-DABA cis/trans mix

750 EP-G-AGBA mix Palmityloleyl glycyl 2-amino-4-guanylbutyric acid;C₁₆C_(18:1)GlyA(Gua)BA²⁺; EP-G-AGBA cis/trans mix

751 JA94881 N′,N′-dimyristyl-α,γ-diaminobutyryl-glycinamide

752 JA94882 N′,N′-dimirystyl-N-γ-carboxamidine-α,γ-diaminobutyrylglycinamide

753 JA10334 N′,N′-dimirystyl-β-alanylamide

1-10. (canceled)
 11. A cationic lipid having the formula VIII:R₈-L₂-Cholesterol wherein, R₈ is selected from the group consisting ofarginyl, N-Boc arginyl, homoarginyl, N-Boc homoarginyl, ornithine, N-Bocornithine, N-benzyl histidyl, lysyl, N-Boc lysyl, N-methyl arginyl,N-methyl guanidine, guanidine and pyridoxyl: and L₂ is a linkerrepresented by NH, glycine, N-butyldiamine or guanidine.
 12. Thecationic lipid of claim 11, wherein said cationic lipid is Bocarginine-cholesterylamide.
 13. The cationic lipid of claim 11, whereinsaid cationic lipid is N-cholesteryl-pyridoxamine.
 14. The cationiclipid of claim 11, wherein said cationic lipid is3′,5′-Di-palmitoyl-2′-deoxy-2′-(N-carboxamidine-beta-alanineamido)uridine.15. The cationic lipid of claim 11, wherein said cationic lipid islinked to polyethylene glycol (PEG).
 16. The cationic lipid of claim 15,wherein said PEG is between about 2000-5000 daltons inclusive.